The XVI Raisa Gorbacheva Memorial Meeting proceeded at the Pavlov University from 15 to 17 September 2022. Since 1990, we follow the tradition of holding this symposium as established by Prof. Boris V. Afanasyev. The present Meeting is attended by more than 150 clinicians from the numerous centers of hematopoietic stem cell transplantation (HSCT) and hematological clinics from Russia, Armenia, Azerbaijan, Belarus, Kazakhstan.

A number of lectures and reports are presented online by the leading European specialists in HSCT and related fields. Many communications concerned advances in HSCT as a potentially curative approach in the patients with hematologic malignancies, especially, acute myeloid leukemia and aggressive lymphomas, as well as rational usage of targeted drugs able to improve prognosis and outcomes in malignant and non-malignant blood disorders.

Central venous access is a mandatory condition to perform allogeneic bone marrow transplantation and intensive therapy in patients with malignancies. Meanwhile, the central venous catheter (CVC) requires daily nursing care, like antiseptic treatment and changing of adhesive bandages. Painful skin irritation and catheter-associated infections are among the adverse events associated with changing these bandages. In order to reduce the severity of a pain syndrome, skin damage, and development of infectious complications, it is necessary to search and implement new methods of CVC care. ConvatecNiltac is a medical anti-adhesive spray which contains liquid silicone compounds for easier removal of adhesive bandages. Currently, there are no available studies representing its efficacy and safety in CVC care in the patients with hematological diseases.

Patients and methods

A total of 56 patients were included into the study with acute myeloid and lymphoblastic leukemia (n=31), chronic myelo- and lymphocytic leukemia (n=4), multiple myeloma (n=6), aplastic anemia (n=2), myelodysplastic syndrome (n=1), Hodgkin’s lymphoma (n=1), myelofibrosis (n=1). All patients were treated at the Department of Bone Marrow Transplantation of the R.M. Gorbacheva Memorial Research Institute of Pediatric Oncology, Hematology and Transplantology in 2021-2022. Their median age was 44 years (18-66). Self-adhesive bandages “Cosmopor” and “Tegaderm” were used as protective dressings for CVC. The Cosmopor patch was changed daily, the Tegaderm bandage was applied every 6 days. Treatment of the CVC injection site was carried out using 3% hydrogen peroxide, and chlorohexidine bigluconate solution (0.05%). The patients were divided into 2 groups: in the 1^st group (n=20), the ConvatecNiltac spray was used to remove the bandage. In the control group (n=26), EcoBreeze antiseptic solution with isopropanol was used to this purpose. During the hospitalization period, the incidence of skin irritation, inflammation of CVC injection site, as well as incidence of systemic infectious complications was assessed. Statistical analysis was determined by the Chi-square test.

Results

The incidence rates of skin irritation at the patch site were lower in the Niltac spray group compared to the controls (5% and 30.8%, p=0.03). There were no differences found in the incidence of infectious complications (febrile neutropenia (70% and 84.6%, p=0.4), sepsis (35% and 53.8%, p=0.33), as well as cases of inflammation at the CVC injection site (20% and 34.6%, p=0.44)) among the patients of both groups. There were no skin allergic reactions to ConvatecNiltac Spray in either case. Conclusion. Niltac spray for removing of CVC self-adhesive bandages causes significantly reduced incidence of skin irritation, but does not affect the risk of developing infectious complications in patients with malignancy.

Keywords

Niltac, skin irritation, central venous catheter.

The method of hematopoietic stem cells (HSCs) transplantation to immunodeficient mice is an important component of the pre-clinical development of medicinal cell products. The traditional in vivo model used to study human hematopoiesis is NSG mice line, but currently the NBSGW is gaining interest [Adigbli, 2021]. Unlike NSG, NBSGW mice do not require pre-transplant conditioning due to the presence of a mutation of c-Kit, the SCF receptor, providing an advantage for transplanted cells. In the current study, for the first time in Russia, NBSGW line was used to study the engraftment and biodistribution of modified human HSCs after in vitro editing of the CCR5 gene mediated by TALEN nuclease. Our objective was to assess the engraftment and biodistribution after transplantation of hHSCs with CCR5 knockout mediated by TALEN nuclease.

Materials and methods

The production of CD34+ HSCs with CCR5 gene knockout cell product prototype was performed according to described protocol [Shakirova Mol. Ther.2022 30;4S1]; prototype samples were cryopreserved. The procedure of transplantation, observation of animals and biomaterial harvesting was performed in the Almazov National Medical Research Centre animal facility. The study included six NBSGW mice (females) obtained from Jackson Laboratory (USA), the median age was 9(8-10) weeks. Pre-transplant conditioning was not performed. On day 0, three mice were transplanted with CD34+ cells at a dose of 1×10⁶, two mice at a dose of 2×10⁶, one control animal was mock-transplanted. Animals were kept in an SPF environment, under daily supervision, weekly veterinary examination and weight control. On day 56, mice were euthanized: samples of blood, bone marrow (BM) and spleen cells were used for donor chimerism assessment by immunophenotyping (FACS) (7AAD/hCD45). The proportion of CCR5 alleles knock out was analyzed by ddPCR [Mock et al., 2015]. A histological examination of tissue samples of BM, spleen, heart, liver, kidneys, intestines, muscles and skin was performed.

Results

During the observation period, no lethal cases were registered. A weekly weight gain was recorded within the species norm. A chimerism level assessed by FACS was 3.8%-38.7% in BM with the maximum values in animal receiving higher HSC doses. In the blood and spleen, the level of chimerism reached 0.2%-3.8% and 0.5%-1.0%, respectively. The frequency of CCR5 gene knockout in spleen cells was 18.6% (13.4%-21.9%), in BM – 19.8%(5.6%-30.1%), in blood samples the alleles with knockout were not found. The histological structure of the skin and muscles, myocardium, intestine, liver, and kidney samples corresponded to the species norm. In the presented samples of the spleen of all animals, aplasia of the white pulp, extramedullary hematopoiesis were detected without significant differences. The histological structure of BM of the control animal was normal. Animals of the experimental group had foci of hematopoiesis formed by non-species-typical medium- and large-sized cells. Histological examination showed no signs of neoplasia.

Conclusion

In this pilot study, the method of transplantation of human HSCs to NBSGW mice was developed. In the absence of pre-transplant conditioning, the engraftment of human HSCs was demonstrated. The potential for multilinear recovery of hematopoiesis and biodistribution of HSCs after the CCR5 gene editing procedure was confirmed.

Acknowledgement

The authors are appreciated for financial support from Russian Foundation for Fundamental Studies, grant №19-29-04025мк.

Keywords

Transplantation, hematopoietic stem cells, genome editing, TALEN, CCR5, model, NBSGW mice, NSG mice.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative option for patients with high-risk acute myeloid leukemia (AML). Nevertheless, relapse of the disease develops in 30-40% of recipients. In recent years, combined immunotherapy with hypomethylating agents and donor lymphocyte infusions (DLI) has shown promising results. The effectiveness of this therapy has not been studied enough in children with AML. Our aim was to study the efficacy and safety of combined therapy with azacitidine and DLI for prophylaxis and relapse prevention after allo-HSCT in children with high-risk AML.

Patients and methods

The study group included 13 children with AML at the median age of 12 years (range, 1 to 7 years). Preventive DLI was performed in 10 patients (78%). Indications for the preventive therapy included evidence for minimal residual disease (MRD) in 6 patients (46%); cytogenetic relapse with mixed chimerism (MC) at 85% detected in one patient (8%); MC levels of 80 to 95% were registered in 3 patients (23%). Prophylactic DLI were performed in 3 patients (23%) due to the following reasons: absence of remission at HSCT (n=1), MRD positivity (n=1) before HSCT, AML evolving from myelodysplastic syndrome (n=1). In 6 patients (46%), allo-HSCT was performed from haploidentical donors; 4 patients (31%) were transplanted from a matched unrelated donor; one patient (8%), from mismatched unrelated donor; HSCT from sibling was performed in 2 cases (15%). Two patients received therapy after 2^nd HSCT. 2 patients developed a clinically significant acute GVHD. Azacitidine therapy was initiated at a median of 124 days (D +42 to +1204) after HSCT. Azacitidine was administered for 5-7 days at a dose of 35-75 mg/m². The median number of azacitidine courses was 3 (1-6). The median number of DLI was 3 (1-8). DLI was administered at the end of azacytidine course. The first median DLI dose was 1.0×10⁵ CD3+ cells/kg (1.0×10⁴-1.0×10⁶), the median total dose comprised 1.5×10⁶ (1.0×10⁴-6.8×10⁷) CD3+ cells/kg. Clinical response to the therapy was defined as achievement of MRD-negative status, and decrease of aberrant transcript copies by 1 log or more, as well as conversion of MC to full donor chimerism (FDC). Overall (OS) and disease-free (RFS) survival were estimated from the time of the first azacitidine administration to death, or last follow-up/relapse, respectively. Kaplan-Meier curves were plotted to estimate OS and RFS levels. The Mann-Whitney U-test was used to compare incidence of response between the groups with different chimerism levels. Statistical analysis was performed using IBM SPSS Statistics v 26 and EZR (Easy R) Free statistical software.

Results

At the median follow up of 12 months, OS was 69%, RFS was 62%. The incidence of response in patients with MRD-positive status reached 83%. Among patients who received preventive therapy, the incidence of response was higher in patients with full donor chimerism, i.e., 83% vs 0%, p=0.014. At the same time, RFS values were similar between these groups: 75% vs 50%, p=0.6. Three patients died from the leukemia progression (23%), one patient (7.6%) deceased from severe graft hypofunction. One patient developed grade III toxic hepatitis during the first course of azacitidine. Therefore, the second course of therapy was carried out at a reduced dose. Chronic GVHD was diagnosed in two patients (15%), i.e., with moderate-grade skin GVHD, and with severe lung GVHD in the patient with a history of severe skin GVHD. We did not observe acute GVHD after combination therapy. The patients received effective combined immunosuppressive therapy with JAK2 inhibitor and tyrosine kinase inhibitors.

Conclusions

Combination therapy with azacitidine and DLI proved to be effective in AML children with high risk for relapse after allo-HSCT. The best response to therapy was achieved in patients with MRD and full donor chimerism. This therapy did not increase risk of developing toxic complications, and may be performed in outpatient setting.

Keywords

Acute myeloid leukemia, azacytidine, donor lymphocyte infusions.

Mature T/NK-cell lymphomas (TCL) are a group of rare, predominantly aggressive non-Hodgkin’s lymphomas that are also characterized by the development of a refractory or relapsed disease during standard chemotherapy-based treatments. The introduction of new treatments, such as targeted immunotherapy, may help to achieve remission in some cases, but not a cure. The only method with a proven curative potential is allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the results of allo-HSCT performed in several centers in Russia and Kazakhstan.

Patients and methods

Allo-HSCT data were analyzed for 24 patients with TCL, who were treated in 7 clinical centers in Russia and Kazakhstan from 2005 to 2022. Histological subtypes included: peripheral T-cell lymphoma, NOS (n=9); ALK-positive anaplastic large cell lymphoma (n=10); nodal TFH cell lymphoma, angioimmunoblastic type (n=2) (WHO Classification, 2022). The remaining patients (n=3) have other rarer types of TCL. The median age was 37 years (1-58). Median number of lines prior to allo-HSCT was 3 (1-9). Pre-transplant treatment was mainly based on targeted immunotherapy (n=14): brentuximab vedotin (n=4); brentuximab vedotin+bendamustine (n=3); nivolumab (n=1); nivolumab+bendamustine (n=1); ceritinib/crizotinib (n=2); brentuximab vedotin + crizotinib (n=1); lenalidomide (n=1); lenalidomide+romidepsin (n=1). The conditioning regimens used were reduced intensity regimens (FluBe n=22; FluMel n=1; FluCy n=1). In all patients, the GVHD prophylaxis regimen was based on posttransplant cyclophosphamide.

Results

18 patients were alive at the time of analysis. Median follow-up was 27 months. The median overall survival (OS) was not reached, the 3-year OS rate was 72%. The median progression-free survival (PFS) was 17 months, 3-year PFS is 50%. The subgroup analysis showed that patients who underwent allo-HSCT in a complete response (CR) have an advantage over patients who underwent allo-HSCT with a partial response or with an active disease status (SD/PD) – (PFS 3-year 73% vs 20% vs 0%, p=0.019). The incidence of acute GVHD grade II-IV and severe GVHD grade III-IV was 25% and 21%, respectively. The frequency of chronic GVHD was 26%. 6 patients underwent anti-relapse treatment after allo-HSCT with CR achieved in 5 patients, these responses persist at the time of follow up. Treatment in the post-transplant period was based on stimulation of the graft-versus-lymphoma (GVL) effects, i.e., donor lymphocyte infusions (n=3), nivolumab (n=1), or targeted treatment, e.g., brentuximab vedotin (n=2); crizotinib/ceritinib (n=3).

Conclusions

Our data provide additional evidence that allo-HSCT is an effective treatment option for patients with TCL. Conducting allo-HSCT in CR is associated with a better prognosis for patients with TCL. In our analysis, we also saw the feasibility of treating relapses after allo-HSCT using targeted antitumor effects and/or stimulation of GVL effects.

Keywords

T cell lymphoma, peripheral, diagnosis, therapy, hematopoietic stem cell transplantation.

Asciminib is a STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor that has shown potential efficacy and a good safety profile according to the results of a phase I and III studies in patients with Ph-positive leukemia failing prior TKIs. In Russia, asciminib is available under the Managed Access Program (MAP) approved by Novartis. While pre-transplant use of 2^nd generation TKIs (nilotinib/ dasatinib) does not change the adverse events rate in allogeneic hemopoietic stem cell transplantation (allo-HSCT) recipients (Niederwieser C., 2021, Masouridi-Levrat S., 2021), there is yet no data available for patients receiving asciminib. This study was aimed to evaluate the safety and effectiveness of pre- and post-transplant aciminib in allo-HSCT candidates.

Patients and methods

From April 2021 to April 2022, 11 patients (pts) with CML were enrolled in the MAP program. Six pts who received asciminib underwent allo-HSCT. Before allo-HSCT, 4 pts achieved a complete hematological response, 1 and 1 MMR and CMR, respectively. All pts received allo-HSCT with reduced dose intensity conditioning regimen including fludarabine 30 mg/m², busulfan 8-12 mg/kg. GVHD prophylaxis was PtCyTxMMF regimen, or monoCy (in case of bone marrow grafting from matched related donor). In 3 cases, (50%), bone marrow (n=1), or PBSC (n=2) from MRD were used. In 3 cases (50%) we used PBSC from unmatched (9/10, 8/10) unrelated donor.

Results

The median patients’ age was 36 (28-52) years, 84% were males. The median time from CML diagnosis to asciminib therapy initiation was 5 (1-15) years. One patient was in chronic phase (CP), 3 in accelerated phase (AP), and 2 had blast crisis (BC), respectively. Five (84%) pts had BCR/ABL1 mutations, 4 pts (66%) had BCR/ABL1^t315i genotype. One patient (16%) had additional chromosomal abnormalities. Four (66%) pts received ≥3 TKIs, 1 (16%) had a history of ponatinib treatment. In 2 pts (34%), the initial dose of the drug was 40 mg BID, 4 (66%) pts started with 200 mg BID. Five (84%) patients did not develop adverse events (AEs) of any grade and 1 (16%) developed 3-4 AEs, however being able to continue the treatment. No differences in toxicity were found between the doses of 80 and 400 mg/day. No toxic events associated with conditioning regimen were registered. One patient developed VOD. The median time of engraftment was D+19 (18-21). In the post-transplant period, 2 pts continued to receive asciminib on D+30/D+60, with development of liver aGVHD after 30 days of treatment, which did not require correction of the underlying immunosuppressive therapy. One patient developed stage 3 intestinal aGVHD requiring glucocorticosteroids and ruxolitinib, resolving at 2 weeks.

Conclusion

Asciminib is potentially effective as bridge therapy prior to allo-HSCT. In patients with advanced-phase disease, asciminib is an optimal drug to improve the disease status before allo-HSCT. More extensive data obtained on larger cohort are needed, in order to assess its impact on long-term survival.

Keywords

Chronic myeloid leukemia, allo-HSCT, tyrosine kinase inhibitors, asciminib.

Cytokine release syndrome (CRS) is a complex of symptoms that can occur during severe infections, as a side effect of immunotherapy and allogeneic stem cell transplantation (ASCT). CRS is usually characterized by fever, vasodilatation and in severe cases can cause macrophage activation syndrome (MAS), multiple organ failure and death. The number of studies describing the impact of CRS on the outcomes of ASCT are limited. The aim of current study is to describe clinical features and significance for prognosis of CRS in patients after ASCT.

Patients and methods

The data was collected retrospectively by analysis of medical histories. Chi-square test and Mann-Whiteney test were used to describe statistical differences between groups. Survival analysis was conducted using Kaplan-Meier method and log-rank test. To describe cumulative incidence of GVHD and relapse, we used competing risk analysis and Grey test. The study enrolled 386 adult patients with acute leukemia who underwent allogeneic stem cell transplantation from January 2015 till December 2021 including 65 cases (16.8%) from matched related donors (MRD); 242 (62.7%), from matched unrelated donors (MUD), and 79 (20.5%), from haploidentical donors. 202 ASCT recipients were diagnosed with AML (52.3%); 184 (47.7%), with ALL. GVHD prophylaxis proceeded with PTBe (n=22, 5.7%), PTCy (n=340, 88.1%). 12 patients (3.1%) underwent TCR ab depletion. The CRS was defined as a fever occurring over the first 3 days after the transplant transfusion, without any signs of infection.

Results

CRS occurred in 16.8% of cases (n=65), with 12.4% (n=48) of grade 1-2. CRS of 3-4^th grade was observed in 4.4% of cases (n=17). CRS was documented more frequently after HaploSCT in 62.0% (n=49); after MRD, in 2 cases (3.1%); after MUD SCT, in 5.79% (n=14) of the cases (p<0.001). CRS mostly presented after ASCT of peripheral blood stem cells (19.3%, n=61), than after bone marrow grafts (5.71%, n=4) (p<0.001). The following laboratory abnormalities were observed more frequently in CRS group vs controls: median ferritin level was 3354 (132-135000) vs 1145 (351-40120), p<0.001; AST, 53 (13-1498) vs 29 (7-1004), p<0.001; ALT, 64 (7-1300) vs 36 (4.6-925), p<0.001; CRP, 128.6 (10.3-509) vs 30.5 (0.5-636), p<0,001; LDH, 241.7 (114-2253) vs 206.8 (91-3597), p<0.001. The median value of H-score (probability of MAS) was higher in CRS group: 77 (42-195) vs 61 (42-175), p<0.001, however, with relatively low specificity scores. Age, gender and conditioning intensity were not associated with CRS incidence. One-year overall survival in the CRS group was 45% (CI95% 30-59%) being 76% in the control group (CI95% 69-79%). Graft failure was also associated with CRS: 12.3% (n=8) vs 4% (n=13), p=0.014. Cumulative incidence of grade II-IV aGVHD and grade III-IV GVHD by the day +125 in CRS group versus non-CRS patients was as follows: 37.1% (CI95% 25.4-48.8%) vs 12.1% (CI95% 8-16%), p<0.001; 26.4% (CI95% 16.3-37.6%) vs 6.3% (CI95% 4-9%), p<0.001; cGVHD incidence was 22% (CI95% 11-34%) vs 22.4% (CI95% 18-27%) p=0.77.

Conclusions

The study demonstrated that existing H-score at CRS scale has low specificity at early post-allograft terms. Significant elevation of ALT, AST, CRP, LDH and ferritin levels was documented in CRS patients. CRS negatively affects overall survival through high incidence of aGVHD and graft failure.

Keywords

Cytokine release syndrome, allogenic stem cell transplantation, acute graft-versus-host disease.

Detection of KMT2A (MLL) gene translocations in the diagnostics of acute myeloid leukemia (AML) is a mandatory procedure due to the high incidence of these aberrations as well as their impact on the patients stratification by risk groups. t(10;11) is among common chromosomal rearrangements after t(9;11). Its occurrence may be up to 8-9% in the AML structure (D. Steinhilber et al., 2018; T. Ksiazek et al., 2020). The main molecular product of t(10;11) is KMT2A-MLLT10 (MLL-AF10), although other gene products have been described. The chimeric KMT2A-MLLT10 transcript is produced by a variety of mechanisms (Klaus et al., 2003). The diagnostics of this translocation is difficult because of cryptic translocations that are not detected by cytogenetic tests or molecular genetics. This study presents four cases of t(10;11)(p12;q23) with the KMT2A-MLLT10 in the children with acute myeloid leukemia (AML) exhibiting different mechanism of its formation, including one case of cryptic translocation.

Materials and methods

The study includes four children with AML M5, whose median age was 11.3 months. RNA was isolated from the bone marrow (BM) mononuclear cells of the patients. The chimeric transcripts of KMT2A gene (AF4, AF6, AF9, MLLT10, ENL, ELL) were discerned by reverse-transcription PCR (RT-PCR) then being verified by direct Sanger sequencing (A. Andersson et al., 2001). Karyotyping of tumor cells was performed using differential G-staining of preparations from the 24-hour BM cell culture (RPMI-1640 supplemented with 15% fetal calf serum, 1% L-glutamine, 1% antimycotic antibiotic). FISH analysis was performed in interphase nuclei and metaphase plates using a locus-specific two-color DNA probe for the KMT2A gene (11q23) (LSI Dual-Color, Break Apart Rearrangement Probe, Vysis, USA), according to the manufacturer instructions. Analysis and registration of data were carried out in accordance with Guidelines of the International System for Human Cytogenic Nomenclature (ISCN).

Results

103 patients with primary AML were diagnosed at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology over the period of 2010 to 2021. KMT2A gene rearrangements were detected in 14.6% of patients. KMT2A-MLLT10 was found in 26.6% of KMT2A-positive patients, and in 3.8% of all examined AML patients. Patient #1 (a boy, 4 months) had a complex rearrangement with the visible marker chromosome formed by fusion of chromosomes 10, 11 and 17, with the KMT2A gene involved. The classic fusion product was detected at molecular level, and its sequencing demonstrated a fusion of exon 10 in KMT2A, and exon 9 of MLLT10. The clone of leukemic cells with t(10;11) translocation and inv(11)(q21q23)/KMT2A(+) inversion resulting into the chimeric KMT2A-MLLT10 gene was detected in patient #2 (a girl, 13.2 months). Of note, this rearrangement is the most common. In patient #3 (a girl, 16 years old), standard karyotyping revealed a complex karyotype with unbalanced translocation variant t(10;11), in which only a derivative of chromosome 10 was detected (der(10)t(10;11)(p12;q23q21)amp(5`KMT2A). RT-PCR revealed the KMT2A-MLLT10 product which involved exon 8 of the KMT2A gene and exon 9 of the MLLT10 gene. Cytogenetic analysis did not reveal chromosomal rearrangements associated with acute leukemia in patient #4 (girl, 9 months). At the same time, the KMT2A-MLLT10 chimeric transcript was detected by RT-PCR. Hence, similar molecular products have been detected in all patients, despite sufficient differences at the chromosomal level.

Conclusion

Thorough identification of rearrangements involving KMT2A gene is extremely important for the disease prognosis, and may succeed only by using a combination of standard karyotyping (G-staining) and molecular cytogenetic techniques (FISH and PCR).

Keywords

Acute myeloid leukemia, diagnostics, KMT2A-MLLT10.

Acute myeloid leukemia (AML) with FLT3 mutations is associated as subtype of AML with higher relapse rate, shorter remission period, decreased survival. Mutations in FLT3 can be considered as a promising molecular target for the treatment of patients with FLT3-mutated AML, particularly refractory or relapsed (r/r) AML. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) as consolidation of second remission improves three-year overall survival from less than 10% to 50%. Our aim was to evaluate the outcomes after gilteritinib therapy in adult pts with r/r AML.

Patients and methods

52 pts with median age 54 (18-79) years were included in the analysis, and 17 of pts were older 60 years old. Status of AML before therapy was as follows: primary refractory – 15 (28.8%), secondary-refractory – 5 (9.6%) and relapsed – 32 (61.5%). Pts with secondary AML were observed in 19.2%. The ITD mutation was detected in 88.5% and TKD – mutation in 11.5% of cases. Patients received gilteritinib 120 mg once daily, mostly 1 or 2 of the 28-day cycles of therapy (59.6% and 25%, respectively).

Results

Overall response (OR) rate was in 41 (78.8%) pts: complete remission (CR) was documented in 32.7%, remission with incomplete recovery (CRi/r) 23.1% and morphological leukemia-free state were also observed in 23.1%. Allo-HSCT was subsequently performed in 28.8% of all treated patients. The median age of this group of patients was 39 (18-68) years. The median time from OR after gilteritinib to allo-HSCT was 45 (8-156) days. Median follow up was 16.5 (7.0-26) months. Median of two-year overall survival (OS) was 6.9 ((95% CI: 5.2-14.9) months and disease-free survival (DFS) in the group of patients who achieved remission was 19 (95% CI: 8,4 – ∞) months. By the multivariate analysis, event-free survival was significantly associated with successful bridging to allo-HSCT (HR=0.3; CI 95%: 0.1-0.7; p=0.08). The most common complication was hematological toxicity: neutropenia 4 gr. – 78% with median duration 36 (4-325) days and thrombocytopenia 4 gr. – 56.1% with median duration was 55 (4-325). Sepsis/bacteremia was observed in 31.8% and bacterial pneumonia – 13.6%. Of the specific complications of Gilteritinib, the most frequently observed were edema (15.2%), myalgia/arthralgia (15.2%), dry skin/itching (12.1%). Therapy-related mortality: No unexpected toxicity was observed after therapy. Therapy-related mortality was 3.8% (2 causes – infections).

Conclusions

Gilteritinib in patients with r/r FLT3-mutated AML demonstrated favorable outcomes with satisfactory tolerance to therapy. Thus, gilteritinib can be used as a bridge to allo-HSCT in adult patients with r/r FLT3-mutated AML.

Keywords

Acute myeloid leukemia, target therapy, gilteritinib.

Ph-like acute lymphoblastic leukemia (ALL) is a subtype of B-cell ALL with poor prognosis. Its prevalence in Russian population is unknown, mainly due to diagnostic challenges. At the same time, there is no standard approach to the diagnosis, and most diagnostic methods used are not financially and/or technically available in clinical laboratories. Moreover, an optimal treatment strategy, as well as relative role of JAK-inhibitors/tyrosine kinase inhibitors (TKI) and allo-HSCT is not defined so far. Here we present the screening diagnostic algorithm and description of examined and treated patients.

Materials and methods

Screening for Ph-like ALL is costly and time-consuming when applying direct search for distinct translocations with specific rearrangements and affected partner genes. Due to this reason, the screening algorithm used in our laboratory is based on determination of the signaling pathways involved in a particular case, in order to choose targeted therapy (TKI/JAK-inhibitors). The algorithm is based on measurement of TSLP levels on cell-surface of B-lymphoblasts using multicolor flow cytometry followed by screening for gene rearrangements by DNA-specific FISH probes (ABL1, ABL2, CSF1R, PDGFRB, JAK2, CRLF2), or qPCR (CRLF2). Sixty-five patients in active B-ALL disease at a median age of 20 (range 2-78 years), without exclusion criteria, were prospectively screened for Ph-like ALL from February 2020 to August 2022 according to the described diagnostic algorithm at the RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation.

Results and discussion

Most patients were examined in ≥1 relapse (n=37, 57%). According to the applied diagnostic algorithm, the incidence of Ph-like B-ALL was 21 of 65 cases (32.2%). The proportion of Ph-like patients in adult group (n=42) was 38%, versus 22% in pediatric group. CRLF2 gene rearrangement was the most frequent finding in the Ph-like cases (n=14, 66.6%), the non-CRLF2 group accounted for 10.7%. IKZF1 gene deletion was detected in 29% of Ph-like cases (n=6). Two patients with ABL1r and ABL2r were treated with dasatinib combined with chemotherapy/blinatumomab resulting into complete remission (CR). In 5 patients with CRLF2r, ruxolitinib was added to chemotherapy/blinatumomab/inotuzumab ozogamicin. Among them, 2 patients did not achieve CR and developed disease progression despite the combination therapy. Seven patients underwent allo-HSCT, only one of them remains in CR1. For the identified Ph-like group, the 5-year OS and RFS rates after ALL diagnosis were 52.2% (95%CI: 19.08%-85.32%), and 33.3% (95%CI: 6.65%-59.95%), respectively.

Conclusion

Diagnosis of Ph-like ALL may not rely on the definite gene expression profile, but rather on detecting aberrations of genes encoding the signaling pathways. A panel of FISH probes covering the most common translocations may be employed as a screening tool to define this unfavorable ALL subgroup. Noteworthy, higher detection of Ph-like cases in our adult cohort compared with literature data could result from selection of relapsed/refractory ALL cases in our center. At the same time, our limited clinical experience demonstrated potential resistance even to the combination therapy with JAK inhibitors. Despite increased OS levels in this high-risk subgroup of ALL patients due to the new therapeutic options, the high rates of late relapses remain the main challenge for this group of young patients. Apparently, Ph-like patients can benefit from allo-HSCT in CR1, but optimal strategies are yet to be determined.

Keywords

Acute lymphoblastic leukemia, Ph-like, tyrosine kinase inhibitors, ruxolitinib, allogeneic stem cell transplantation.

Outcomes for patients (pts) with refractory/relapsed acute myeloid leukemia (r/rAML) are dramatic because of the low response rate, which reaches less than 30%. Allogeneic hematopoietic stem cell transplantation (HSCT) as consolidation of second remission improves three-year overall survival (OS) from less than 10% to 50%. By this way, the search for new therapeutic options for pts with r/rAML is relevant so far. Our aim was to evaluate the outcomes after high-dose chemotherapy (HDChT) and immunochemotherapy in adult pts with r/rAML.

Patients and methods

92 pts with median age 34 (18-61) years were included in the analysis. Status of AML before therapy was as follows: relapsed (Rel) n=37, refractory (Ref) n=55. HDChT group included 40 pts treated with “FLAG” with or without anthracyclines – “FLAG” n=17 or “FLAG-Ida/Mito” n=23; another group of 52 pts received “FLAG” with gemtuzumab ozogamicin – “GO-FLAG”. The groups were comparable for age (p=0.56), sex (p=0.31), genetic risk ELN2017 (p=0.97) and AML status (p=0.09). Special feature of “GO-FLAG” pts group were extramedullary disease (ED) in 16 (31%) cases.

Results

Overall response (OR) after HDChT was achieved in 25 (62.5%) pts: complete remission (CR) – 50%, CR with incomplete recovery (CRi/r) – 12.5%. OR after “GO-FLAG” therapy were in 38 (73.1%) pts: CR – 51.9%, CRi/r – 21.2. The less rate OR were observed after “FLAG” 41.2% vs “FLAG-Ida/Mito” 78% (18 out of 23) vs “GO-FLAG” 73.1%, p=0.025. HSCT after HDChT was performed in 25 pts (62.5%), 19 pts were in remission and median time from OR to HSCT was 89 (19-336) days. Subsequent HSCT in the “GO-FLAG” group was performed in 27 pts (51.9%) and 25 pts were in remission, with median time to HSCT was 40 (16-224) days. Median follow-up was 35 (18.4-52.2) months. Two-year OS in the HDChT and “GO-FLAG” groups was 8.7 (95% CI: 5.3 to 16.7) months and 6.4 (95% CI: 4.2 to 8.8) months, respectively (p=0.38). Two-year relapse-free survival: after HDChT-8.5 (95% CI: 4.3-18.3) months, “GO-FLAG”-5.1 (95% CI: 3.0-15.5) months, p=0.08. According to multivariable analysis, the risk of any event (EFS) was decreased after HSCT (HR 0.27; CI95%: 0.19-0.1; p<0.001), after “FLAG-Ida/Mito” (HR 0.2; CI95%: 0.09-0.46; p<0.001) and after “GO-FLAG” (HR 0.23; CI95%: 0.1-0.53; p<0.001), and increased with adverse genetic abnormalities (HR 2.4; CI95%: 1.0-5.5; p=0.048). Analysis of toxicity demonstrated no differences depending on the therapeutic groups. Therapy-related mortality: after HDChT – 10% (causes in 3 pts – cerebral hemorrhagic, 1 pts – sepsis); “GO-FLAG” – 9.6% (3 pts – infections, 1 pts – cerebral hemorrhagic, 1 pts – progression).

Conclusions

The “FLAG”, “FLAG-Ida/Mito”, and “GO-FLAG” demonstrated favorable outcomes in pts with r/rAML. After “FLAG-Ida/Mito” and «GO-FLAG» therapy, there was significant increase in remission rates when compared to “FLAG”. A distinctive feature of “GO-FLAG” was the high response rate in patients with ED. The main predictors favorably influencing EFS were: the choice of “FLAG-Ida” and “GO-FLAG”, subsequent HSCT.

Keywords

Acute myeloid leukemia, target therapy, high-dose chemotherapy, gemtuzumab ozogamicin.

Currently, the population data concerning treatment efficacy in adult patients with acute myeloid leukemia (AML) are virtually absent in Russian Federation. Moreover, according to some limited observation series, the survival rate for this disorder does not exceed 10-20%. To determine population characteristics and treatment results of AML patients in Russia, a Cooperative Program for the Diagnosis and Treatment of Acute Myeloid Leukemia in Russian Federation was initiated by the Tin’kov Family Foundation. The key parameters of the program include early immunophenotypic and molecular, as well as cytogenetic diagnostics and HLA typing, providing early access to allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the risk groups with intermediate and unfavorable genetic findings.

Patients and methods

This analysis was performed in an initial cohort of 200 patients enrolled from 29 participating centers since October 2021 to August 2022. After signing an informed consent form and evaluating compliance with inclusion criteria, the patients were subject to routine diagnostics, as well as to karyotyping, immunophenotyping, molecular genetic analyses of the bone marrow. HLA typing of the patients and potential donors was performed at the facilities of RM Gorbacheva Research Institute.

Results

The primary diagnosis of AML was confirmed in 82.5% of patients (n=165). Acute lymphoblastic leukemia was diagnosed in 6.5% (n=13); acute promyelocytic leukemia, in 4.5% (n=9); myelodysplastic syndrome, in 3.5% (n=7), and other diagnoses were established in 3% of cases (n=6). Further analysis was performed in the verified AML group. The median follow-up time for the living patients was 54.5 (18-307) days. The female-to-male ratio was 53.9% versus 46.1% (89 to 76 cases, respectively), with median age of 52 years old (18 to 83). When stratified by the ELN criteria, favorable risk was defined in 20% of patients (n=33), intermediate, in 50.3% (n=83); unfavorable, in 29.7% (n=49). AML with myelo-dysplasia-related changes was diagnosed in 18.8% (n=31) of patients, and 6.7% (n=11) have undergone prior chemotherapy due to another malignancies. According to the results of cytogenetic examination (n=114), aberrant karyotypes were found in 56% (n=64), complex karyotype was detected in 11.4% of cases (n=13). According to results of molecular genetic analysis, mutations were most often detected in FLT3 (35/141, 24.8%) and NPM1 (30/139, 21.6%) genes. In the group of patients who underwent induction therapy (n=110), complete remission was achieved in 47% (n=52) of cases, whereas 16% of the patients (n=17) were primarily refractory, early mortality was registered in 37% (n=41). In the group of patients who underwent allo-HSCT (n=12), the median time from the time of diagnosis to HSCT was 189 days (104 to 266). Allo-HSCT from matched related, matched unrelated, mismatched unrelated and haploidentical donors was carried out in 33.3% (n=4), 8.3% (n=1), 41.7% (n=5) and 16.7% (n=2) of patients, respectively. Allo-HSCT was performed in the first complete remission in 66.6% (n=8) of cases; in the state of active disease, in 33.4% (n=4). Early mortality was 12.5% among the patients who achieved remission (n=1), being 75% (n=3) in the active disease group. The median follow-up time after allo-HSCT for survivors was 60 days.

Conclusion

As seen from the first results of the Cooperative Program for the Diagnosis and Treatment of Acute Myeloid Leukemia in the Russian Federation, a large proportion of genetically unfavorable AML forms was revealed. We have also noted high percentage of early mortality and long median time before allo-HSCT were. During further development of the program, one should overcome these major barriers in order to improve the survival rates.

Keywords

Acute myeloid leukemia, allogeneic hematopoietic stem cell transplantation, cooperative program.

Hematopoietic stem cell transplantation (HSCT) has improved the results of treatment in patients with hematological malignancies. This option is included in Russian treating protocols of acute B-lymphoblastic leukaemia (B-ALL) from very high-risk group, in particular for Ph-positive variant. The aim of our work was to determine an importance of HSCT after the achieving of CR1 in adult patients with Ph-positive acute B-lymphoblastic leukaemia, to determine risk factors during an execution of program therapy.

Materials and methods

From 2012 to 2022, 68 patients with first identified Ph-positive acute B-lymphoblastic leukaemia were included in retrospective analysis, median age is 35 y.o. (17-72), m/f=29 (43%)/39 (57%). Leukocytosis more than 30×10^9/л was identified in 29 patients (43%). The type of chimeric transcript was identified in 56 patients: р190/р190+р210/р210=33 (59%)/9 (16%)/14 (25%). Patients were treated according to the research protocols RALL–2009+TKI (12 patients); RALL–2012+TKI (26), and RALL–2012m+TKI (24). An approach of low-dose constant chemotherapy was applied for the program therapy (CT), in combination with non-stop usage of imatinib being replaced by dasatinib in cases of lacking molecular remission on the 70th day of the protocol.

Results

Clinical and hematological remission (CR1) was achieved after 1st induction in 67 (98.5%) patients. Molecular complete remission (MolCR) on the 70th day was achieved in 25 patients (37%), dependent on the protocol used: RALL–2009+TKI/RALL–2012+TKI/RALL–2012m+TKI=4 (33%)/13 (50%)/8 (33%). Allo-HSCT was performed in 31 patients (46%) with CR1. The 5-year overall survival (OS) comprised 55%; relapse-free survival (RFS), 35%; mortality, 35%.; early mortality was 3% (n=2). No statistically significant differences were found for OS and RFS, depending on age, treatment protocol, type of chimeric transcript, achievement of MolCR on 70th day. There were statistically significant differences of OS between patients with leukocytosis >30×10^9/l at the onset (32%), versus cases with lower leukocytosis (74%, р=0.024). Four subgroups of patients were discerned during the OS and RFS analysis: CT with or without allo-HSCT in the patients <45 y.o. (“CT+BMT”, “CT”); CT with, or without allo-HSCT over 45 y.o. («CT45+», “CT+BMT45+”). OS levels in all groups did not show significant differences (“CT+BMT” 68% vs “CT” 50% vs “CT45+” 67% vs “CT+BMT45+” 40%). Better RFS was revealed in the “CT45+” group (70%, р=0.016). RFS in “CT+BMT” group (43%) was significantly higher compared to the «CT» group (0%, р=0.005). There weren’t any significant differences between groups “CT45+” (70%) and “CT+BMT45+” (40%), whereas RFS of “CT45+” reached 70%, RFS of “CT+BMT45+” reached 40%. Transplant-related mortality in “CT+BMT” group was 15% (n=4) (sepsis – 3, graft failure – 1), 12% (n=3) of patients died due to relapse of the disease. Transplant-related mortality in “CT+BMT45+” group (n=5) was 60% (graft failure, 1; graft-versus-host disease, 2 cases).

Conclusion

Allo-HSCT in CR1 improves the results of treatment in patients with Ph-positive acute B-lymphoblastic leukaemia due to reduced probability of relapse. There are controversial results of allo-HSCT in patients over 45 y.o. with CR1, due to high transplant-related mortality.

Keywords

Acute B-lymphoblastic leukaemia, Ph-positive, allo-HSCT.

Relapse of acute myeloid leukemia (AML) after allo-HSCT remains one of the main causes of treatment failure. Classical approaches for predicting the risk of AML recurrence after allo-HSCT are based on detection of minimal residual disease (MRD) by flow cytometry and molecular biology studies of recurrent aberrations. Recent studies of gene characteristic of leukemic stem cells (LSCs) defined at the onset of the disease have shown independent prognostic value for children with AML. Data on the pre-transplant level of LSC expression can serve as an additional tool in assessing the risk of post-transplant AML relapse. The aim of our study was to evaluate the influence of MRD status on the results of allogeneic HSCT (allo-HSCT) in children with AML, being determined both by classical methods and according to the genes characteristic of LSCs.

Materials and methods

To assess the MRD by classical approaches, we analyzed data of 95 children with AML in the 1st and 2nd remission. Allo-HSCT was performed over the period of 2008 to 2021. The median age at the time of allo-HSCT was 8 years (5 months to 19 years). Negative MRD status was documented in 67 (70.6%) patients, 28 (29.4%) children had a positive MRD status according to molecular genetic studies and/or immunophenotyping results. Myeloablative conditioning (MAC) was given to 58 (61%) patients, reduced-intensity conditioning (RIC) was performed in 37 (39%) patients. Allo-HSCT from full-matched related donors was performed in 13 (15%) patients; from unrelated donors, in 48 cases (50%); from haploidentical donors, in 34 (35%) patients. All patients received GVHD prophylaxis including post-transplant cyclophosphamide (PtCy) in 59 cases (68%). For pre-transplant evaluation of the LSC gene expression, RT-PCR was performed for the bone marrow samples of 50 patients. At the time of allo-HSCT, 37 (74%) children with AML were in 1st or 2nd remission, whereas 13 (26%) exhibited active disease. The median age in this cohort was 6 (1-18) years. Among the patients in 1^st or 2^nd AML remissions, 3 children (8%) received allo-HSCT from a full-matched sibling donor; 15 (41%), from an unrelated donor; 19 (51%), from a haploidentical donor. GVHD prophylaxis based on PtCy was received by 29 patients (78%). The DNMT3B, GPR56, CD34, SOCS2, SPINK2, IL2RA, FAM30A, and ABL genes were studied by real-time PCR, followed by calculation of the pLSC6 score using the following formula: (DNMT3b*0.189)+(GPR56*0.054)+(СD34*0.0171)+(SOCS2*0.141)+(SPINK2*0.109)+(FAM30A*0.0516).

Results

At a 5-year median follow-up in MRD+ patients detected by standard methods, the OS is 67.9% vs 73.1% in MRD(-) patients (p=0.83). Relapse-free survival (RFS) was 53.6% vs 80.6%, respectively (p=0.01). When assessing expression levels of the genes characteristic of LSC, 18/37 patients (49%) were assigned the pLSC6 level above the median. Only 6/18 patients in 1^st or 2^nd remission with high pLSC6 were MRD-positive. The linear regression analysis included the patients with pre-transplant response, as well as patients with active disease. It did not show any association between blast counts/MRD and pLSC6 values (OR 1.002; 95% CI: 0.979, 1.025). The 1-year RFS in the CR patients was not significantly different between low-pLSC6 (78.9%) and high-pLSC6 (66.7%) cases (p=0.62). The early relapse rate in CR patients was significantly higher in high-pLSC6 subgroup compared to low-pLSC6 (22% and 0%, accordingly; p=0.03).

Conclusions

Minimal residual disease status before allo-HSCT does not exert a statistically significant effect upon OS. However, MRD-positivity negatively affects RFS values. The pre-transplant level of genes characteristic of LSC showed prognostic significance independent of classical methods for assessing MRD, with respect to early post-transplant AML relapse in children.

Keywords

Acute myeloid leukemia, allogeneic hematopoietic stem cell transplantation, minimal residual disease, leukemic stem cells.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in 20-30% of adult patients with acute myeloid leukemia (AML). Detection of FLT3 mutation is associated with a higher relapse rate (RR) and leads to worse overall (OS) and event-free (EFS) survival. Administration of midostaurin (Mido), a potent FLT3-kinase inhibitor, in AML FLT3+ in combination with chemotherapy (CT) and after allogeneic hematopoietic stem cell transplantation (allo-HSCT) can reduce the relapse rates.

Objective

To evaluate the role of allo-HSCT in adults with FLT3+ AML treated with Mido in CT.

Patients and methods

The study included 74 patients who received CT with Mido in induction, consolidation and maintenance therapy. The median age was 48 (18-74) years. Median follow-up period was 11.7 (1.1-36.8) months. The median level of leukocytes was 50.6 (1.6 to 469.0)×10⁹/L. FLT3-ITD mutation was detected in 61 (82%) patients. The favorable prognostic group ELN2017 (Fav-ELN2017) included 4 (5%) patients; intermediate (Int-ELN2017), 58 (78%) patients; unfavorable prognosis (Unfav-ELN2017) – was assessed in 12 patients (16%).

Results

Complete remission (CR) was achieved in 51 (69%) patients (Fav-ELN2017 – 100%; Int-ELN2017 – 79% and Unfav-ELN2017 – 50%). Early mortality was 8% (6 patients). 17 patients (23%) were primarily refractory. The median duration of CR in Int-ELN2017 was 7.5 (0.5-36) months compared to 4.7 (2.7-35) months in Unfav-ELN2017 group. In the total group, OS was 53 months (95% CI 37-69), and FFS, 37 months (95% CI 23-51). 17(33%) patients developed a relapse at a median of 7 months (2.7-13). The relapse rate was higher in Unfav-ELN2017, 67% compared with 29% in the Int-ELN2017 group. Allo-HSCT was performed in 21(28%) patients, 12 of them were in CR1. After allo-HSCT, Mido was restarted at a median of 75 (31-370) days. The disease-free survival (DFS) was longer when allo-HSCT was performed in CR1, compared with patients without HSCT, i.e., 81 (95% CI 57-99) vs 40 months (95% CI 17-63, p=0.06).

Conclusion

Disease-free survival in adult patients with FLT3+AML is higher after allo-HSCT performed in CR1 compared to the patients who received only CT in combination with Mido.

Keywords

Acute myeloid leukemia, target therapy, midostaurin.

There are only limited treatment options for the patients with relapsed/refractory B-cell Non-Hodgkin Lymphoma (r/r NHL). Tumor cells may exploit the programmed death-1 checkpoint pathway to evade immune surveillance. In the current study, we evaluated efficacy and safety of programmed cell death-1 blockade by nivolumab combined with bendamustine, gemcitabine and rituximab (BeGeRN) in the patients with r/r NHL.

Patients and methods

This prospective study included 42 patients with r/r NHL, among them 52.4% (n=22) with diffuse large B-cell lymphoma (DLBCL); 35.7% (n=15) with primary mediastinal B-cell lymphoma (PMBCL) and 11.9% (n=5) with follicular lymphoma grade 3 (FL). Their median age was 42 (range, 18 to 66) years. Most patients (76.2%, n=32) had a primary chemoresistant disease. The median number of prior therapies was 3 (range, 2-6) lines. Ann Arbor stage III-IV was established in 69% patients (n=29), and bulky disease was documented in 35.7% of cases (n=15). Six patients (14.3%) underwent autologous stem cell transplantation prior to BeGeRN treatment. Median number of BeGeRN cycles was 2 (range, 1-4).

Results

At median follow-up of 20.4 months (range, 2-60), the overall response rate (ORR) in the total group of patients was 41% (n=17), with complete response (CR) in 21% (n=9), and partial response (PR) in 20% of cases (n=8). Median duration of response for all patients with ORR (n=17) was 6.5 months (range: 0.8-59). The ORR value in patients with DLBCL was 45.5% (CR 18.1%), being 40% in PMBCL patients (CR 20%), and with ORR of 75% in the patients with FL (CR 50%). ORR in the patients with bulky disease was 33.3% (CR 20%) compared to ORR of 59.3% (CR 22.2%) in the patients without bulky disease. Four patients who achieved objective clinical response to the therapy underwent hematopoietic stem cell transplantation (auto-HSCT n=2, allo-HSCT n=2). Two patients who received auto-HSCT developed a relapse, and both patients after allo-HSCT remained in CR (up to 60 and 58 months). Three patients with BeGeRN failure received CAR-T cell therapy, and four patients were treated with glofitamab. Two-year overall survival and progression-free survival were 30% and 16%, respectively. Most common adverse events (grade 3-4) were associated with hematological toxicities: anemia, neutropenia, and thrombocytopenia developed in 22%, 62% and 37% patients, respectively. Non-hematologic toxicities grade 3-4 included febrile neutropenia in 36%, autoimmune cytopenia in 2% and Stevens-Johnson syndrome (response to glucocorticosteroids) was observed in 2% of patients.

Conclusions

Immunochemotherapy with nivolumab, bendamustine, gemcitabine and rituximab has a manageable toxicity profile and may lead to objective clinical response in the patients with r/r NHL. However, durability of the response to therapy is not long, and consolidation therapy is required, e.g., allo-HSCT, CAR-T, bispecific antibodies.

Keywords

B-cell non-Hodgkin lymphoma, immunochemotherapy, nivolumab.

High-dose chemotherapy (HDCT) followed by autologous transplantation of hematopoietic stem cells (ASCT) is the gold standard of treatment for patients with primary-refractory and relapsed forms of Hodgkin’s lymphoma (R/R HL). There are several most commonly used conditioning regimens for HDCT followed ASCT. However, there are currently no data on the conduct of randomized studies that would compare the effectiveness and toxicity of different regimens.

Aims

Comparison HDCT (CLV, LEAM, BeEAC) as a conditioning regimen before ASCT for the treatment of R/R HL.

Patients and methods

In retrospective study were included 279 patients with HL, median age 30 years; 121 men and 158 women. All patients received HDCT and ASCT in National Medical and Surgical Center named after N. I. Pirogov (2006-2018). Conditioning regimens: CLV (cyclophosphamide, lomustine, etoposide) – 78 patients, LEAM (lomustine, etoposide, cytarabine, melphalan) – 129 patients, BeEAC (bendamustine, cytarabine, etoposide, cyclophosphamide) – 72 patients.

Results

Efficiency of different conditioning regimens. Comparative analysis of overall survival (OS) showed lower OS rates in patients who received LEAM. 5-year overall survival: CLV – 82%, BeEAC – 78%, LEAM – 70% (p=0.04). 5-year progression-free survival (PFS) were comparable with LEAM (53%), BeEAC (50%) and CLV (50%) (p=0.66). Hematologic toxicity of different regimens (CLV, LEAM, BeEAC): All patients developed grade IV neutropenia, anemia with/without transfusion demands, severe thrombocytopenia with transfusion requirements in most cases. Duration of neutropenia was the same (median of 9 days). Duration of thrombocytopenia in CLV regimen was 9 days, LEAM and BeEAC – 11 days (p=0.03). Anemia Grade II (median) was registered in CLV, Grade III (median) in LEAM and BeEAC regimens (p>0.05). Non-hematologic toxicity was as follows: The incidence of oral mucositis and enteropathy was higher in the BeEAC (40.3%, n=29) and LEAM (56.6%, n=72) regimen compared to the CLV regimen (34.6%, n=27). The development of cardiotoxicity was also more often noted in the BeEAC and LEAM groups, 6.9% and 2.3%, respectively. Indices of liver toxicity, pulmotoxicity and the incidence of infectious complications were comparable. Transplant-related mortality (until D + 30) was: CLV – 1.3%, LEAM – 3.1%, BeEAC – 2.8% (р>0.05). Efficiency of conditioning regimens was assessed as overall survival (OS) and progression-free survival (PFS). Comparative analysis of OS rates showed lower OS values in the patients who received LEAM treatment, with 5-year OS of 82% after CLV, 78% following BeEAC, and 70% after LEAM conditioning (p=0.04). The 5-year progression-free survival rates (PFS) were comparable for the patients treated according to LEAM (53%), BeEAC (50%) and CLV protocols (50%, p=0.66).

Conclusion

HDCT followed by ASCT is the best therapeutic approach for a R/R HL. BeEAC, LEAM and CLV conditioning regimens being considered as viable alternatives. Our results suggest a comparable efficacy of BeEAC, LEAM and CLV conditioning in terms of survival and disease control. However, we also observed higher rates of gastrointestinal and cardiac toxicities in patients transplanted after LEAM and BeEAC. The worst OS in patients received LEAM can be explained by the fact that the regimen was implemented at our hospital earlier than others schedules when such drugs as Brentuximab vedotin and checkpoint inhibitors were not available to the patients with relapse after HDCT and ASCT.

Keywords

Transplantation, AHSCT, conditioning regimens, Hodgkin’s lymphoma, BeEAC, CLV, LEAM.

Marginal zone lymphoma (MZL) is the second most common subtype of indolent B-cell non-Hodgkin’s lymphoma. MZL comprises approximately 6% of all lymphoid malignancies. Based on the World Health Organization data MZL is subdivided into 3 major categories: extranodal MZL (EMZL), MALT, nodal MZL (NMZL), and splenic MZL (SMZL). Due to the lack of established standards of therapy and the limited amount of data on the results of treatment in the Russian Federation, the analysis of the histological, clinical and epidemiological characteristics of MZL is an urgent topic for study. The aim of this work is to analyze the strategy of first-line therapy for patients with MZL in real clinical practice at the Raisa Gorbacheva Memorial Research Institute, Pavlov University.

Patients and methods

A retrospective single center study included 77 patients (25m/52f) with a histologically confirmed diagnosis of MZL. The primary endpoint was overall survival (OS) of patients with MZL, defined as the time from the start of first-line therapy to death from any cause. The secondary endpoint was progression-free survival (PFS), the time from the start of first-line therapy to disease progression, relapse, or death from any cause, PFS was censored at the time of initiation of second-line therapy. In assessing survival, data were censored according to the date of last contact if there was no event at the time of follow-up. Survival were estimated using the Kaplan-Meier method.

Results

The median age of patients was 70 (28-97). Among the morphological subtypes, SMZL occurred in 42% (n=33), MALT in 29% (n=22), NMZL in 29% (n=22). At the time of diagnosis, stage I was observed in 13% (n=10), II – in 5% (n=4), III – in 1% (n=1), IV – in 77% (n=59), in 4% (n=3) no information available. At the time of diagnosis, ECOG0-1 in 87% (n=67), ECOG2-3 in 13% (n=10). B-symptoms were detected in 40% (n=31). FISH was performed in 29% (n=22), chromosomal rearrangements involving the p53 was detected in 23% (n=5). The 1^st line therapy included: R-mono for 64%(n=49), R-CHOP for 12%(n=9), RB for 9% (n=7), surgical treatment for 5% (n=4), CHOP-like regimens for 4%(n=3), other options of the therapy 4% (n=3), observation for 3% (n=2). Maintenance therapy with rituximab was performed in 40% (n=31). Among patients who received conservative treatment, complete response after the first line of therapy was observed in 21% (n=16), partial response in 39% (n=30), disease stabilization in 13% (n=10), progression in 21% (n=16), no data in 6% (n=5). The median follow-up from the start of first-line therapy was 29 months (2-101). Median OS not reached. Three-year PFS for the entire study group – 62.2%. The median progression-free survival was 45 months.

Conclusions

Analysis of clinical characteristics and first-line treatment strategy in patients with MZL in real clinical practice was performed. The basis of MZL therapy is rituximab therapy in combination with various regimens of mono- and polychemotherapy. Patients in the study population had a favorable clinical prognosis. For reliable identification of the dependence of outcomes and the chosen treatment regimen, it is necessary to increase the number of patients and the duration of observation.

Keywords

Marginal zone lymphoma, first-line therapy.

Despite the widespread use of antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection is associated with an increased incidence of non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Concurrently, autologous stem cell transplantation (ASCT) becomes a feasible approach to either rescue or consolidate HIV-related lymphoma patients. However, there is only limited number of prospective matched case-controlled studies to prove the safety and efficacy of ASCT in HIV-related lymphoma.

Patients and methods

Between August 2014 and May 2022, fifteen patients with HIV-related lymphoma who underwent ASCT were included in prospective matched case-control study (study group, n=15). Sixty non-HIV-infected patients were enrolled into the control group (n=60, 1:4). Their median age was 35 (19-66) y.o. The underlying diseases in study group were as follows: 7 cases of HL (46.6%), and 8 patients with NHL (53.4%), with complete remission prior to ASCT (73.3%). Conditioning regimen was BEAM with BCNU replaced by Bendamustine (160 mg/m²/day on D-7, D-6). HIV viral load was undetectable, and the median number of CD4+ cells was 360 cells/mcL (133-715). All patients received HAART schedules. The median follow-up time was 2.9 years (1 day to 5.2 years). The primary endpoints were, as follows, overall survival (OS), progression-free survival (PFS) and time-to-progression (TTP) 2 years after ASCT. Secondary endpoints included terms of hematopoietic recovery, organ toxicity and transplant-related mortality (TRM). Common Terminology Criteria for Adverse Events (CTCAE 5.0) were used for the toxicity analysis.

Results

The 3-year OS (n=75) was 88%: 86.7% in the study group, 88.3% among control group, and did not significantly differ between the groups (p= 0.876). Progression-free survival (PFS) at 3 years was 66.7% in the study group, and was not different against the control group (76.7%, p=0.411). Time-to-progression (TTP) at 3 years was 20% in study group, versus 18.3% in controls (p=0.796). Complete remission at the time of ASCT was associated with better PFS levels (p=0.049) and TTP (p=0.052) in the total group. The median recovery terms for leukocytes, neutrophils, and platelets were, respectively, D+16 (10-25), D+15 (12-30), and D+15 (11-31) in study group compared with D+15 (10-22), D+14 (10-23), and D+14 (8-31) in control group. There was no intergroup difference in the rates of organ toxicities, according to CTCAE criteria.

Conclusion

Three-year overall survival in the patients with HIV-related lymphoma was 86.7%; PFS, 66.7%, and TTP, 20%. Complete remission at the moment of ASCT improved PFS and TTP levels in the total group. Our data provide further evidence that ASCT is a safe and effective option for the patients with HIV-related lymphoma.

Keywords

Hematopoietic stem cell transplantation, autologous, lymphoma, HIV-related.

Autologous transplantation of peripheral hematopoietic stem cells (PHSC) significantly improves survival rates. Autologous PHSC transplants are included into standard therapy schedule of the patients with multiple myeloma (MM). The harvested transplants should meet the generally accepted quality criteria, in order to implement appropriate transplantation program. High-dose cytostatic therapy followed by PHSC transplantation is known to be performed after induction treatment. The risk of collecting low-quality PHSC transplants increases if the patients underwent several lines of therapy including melphalan, lenalidomide, fludarabine, and irradiation courses. This category of patients is referred to the group of “poor mobilizers”. To improve the quality of transplant preparations, a selective inhibitor of CXCR4 – plerixafor is used which blocks the CXCL12 ligand binding by inhibiting CXCR4, thus promoting release of PHSC with an ideal immunological profile: CD34+CD133+CD90+CD38-CD45+ from the bone marrow. The purpose of this study was to perform quality evaluation of autologous PHSC transplants obtained from MM patients with plerixafor used in the mobilization regimen.

Materials and methods

The study included 13 patients diagnosed with MM referred to the group of “poor mobilizers”, or with history of failure to mobilize PHSC using standard techniques. The treatment schedule was as follows: since the 1st day of mobilization, injections of granulocyte colony-stimulating factor (G-CSF) were started at a dose of 12 μg/kg/day. Daily monitoring of CD45+/CD34+ cells was performed by means of flow cytometry (BD FACS Canto II). In the absence of target PHSC levels, plerixafor was administered on the 4th day (0.24 μg/kg), thus enabling initiation of apheresis on the next day.

Results

The number of collected CD34+/CD45+ cells averaged 5.3×10⁶/kg at the first apheresis, 8.3×10⁶/kg in the second session, thus being sufficient to perform two transplantations at the optimal cell numbers. Immunological tests (CD34+/CD45+ cell counts and cultural assays (assessment of CFU numbers) were used to assess the quality of the harvested transplant after cryostorage. All patients underwent double tandem courses of high-dose cytostatic therapy with autologous PHSC transplantation using conditioning regimen with melphalan (200 mg/m², or 140 mg/m² in the patients >65 years). Due to pegfilgrastim injections early post-transplant, we preferred platelet restoration in order to assess the engraftment terms. On day +13, the level of granulocytes in all patients was more than 0,5*10³ /µL, the level of platelets exceeded 20*10³ /µL (without transfusion support).

Conclusions

Hence, our experience has shown that the addition of plerixafor to the standard mobilization schedules in the patients with a history of collection failure or poor prognosis for mobilization makes it possible to obtain a PHSC leukoconcentrate that meets all quality standards in terms of optimal amount of hematopoietic cells for two transplants.

Keywords

Hematopoietic stem cells, mobilization, multiple myeloma, plerixafor.

Nivolumab (Nivo) showed its efficacy in patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL) in clinical trials. Continued accumulation of the long-term experience in PD-1 inhibitors treatment is required to support previous data. Our aim was to evaluate long-term data on the efficacy and safety of Nivo therapy in patients with r/r cHL.

Materials and methods

A retrospective cohort of 182 patients was analyzed: 36% of patients (n=66) were treated with Nivo (40 mg); 64% (n=116) were treated with Nivo at a dose of 3 mg/kg in the RM Gorbacheva Research Institute and Regional Oncological Clinic in Irkutsk. Overall response rate (ORR), progression-free survival (PFS), overall survival (OS) were analyzed. PFS was censored upon initiation of additional therapy. The response was evaluated every 3 mo by PET-CT using LYRIC criteria. Adverse events (AE) were analyzed by NCI CTCAE 5.0.

Results

Patient characteristics included demographic parameters, data on previous therapy, chemoresistance, relapse rates, and disease stage at the time of Nivo initiation. Median follow-up was 54.9 mo (0.5-77.7). ORR was 68%, e.g., complete response (CR), 33%; partial response, 35%; stable disease, 7%; indeterminate response, 18%; progressive disease, 7% of cases. Median OS was not achieved, 5-year OS was 94.3% [95% CI: NA-NA]; median PFS was 19.2 mo [95% CI: 15.4-23.5]; 5-year PFS was 25.6% [95% CI: 17.5-34.6]. Several factors, including patients’ age, Nivo dose, number of previous therapy lines, prior auto-HSCT or brentuximab vedotin (BV) treatment have not had a statistically significant effect upon PFS. However, B-symptoms at Nivo initiation (median PFS, 35.6 mo [95% CI: 19.1-NA] vs 15.3 mo [95% CI: 11.5-19.3], p=0.0006); gender (median PFS in females was 23.4 [95% CI: 20.1-26.8] vs 13.7 in males [95% CI: 8.8-18.7], p=0.018), and early CR after 3 mo of Nivo therapy (median PFS 32 mo [95% CI: 20.4-NA] vs 17.9 mo [95% CI: 13.7-21.7], p=0.008) were significantly associated with PFS. Any grade of AE was detected in 72% of analyzed patients (n=173), including gr 3-4 AE in 21% of cases, and gr 5 AE in 2% of pts (2 secondary MDS, and one case of pneumonia). Additional therapy after Nivo was required in 78% of patients and included, e.g., Nivo monotherapy in 25%, chemotherapy (CT) in 7%, BV monotherapy in 7%, Nivo combined with CT or BV in 57%, allo- and auto-HSCT performed in 3% and 1% of patients respectively. Allo-HSCT was carried out in a total of 15% of patients after initial Nivo monotherapy, either immediately, or after additional therapy.

Conclusion

The obtained data demonstrate high efficacy and acceptable toxicity profile of Nivo therapy in the patients with r/r cHL. Such prognostic factors as absence of B symptoms at the time of Nivo initiation, female gender and early CR achievement were significantly associated with increased PFS duration. Since the Nivo therapy does not allow curation of the disease in most patients, some additional therapy after Nivo, including PD1 inhibitors, as well as allo-HSCT, may increase the survival rate in this group of patients.

Keywords

Nivolumab, PD-1 inhibitors, immunotherapy, Hodgkin lymphoma, LYRIC criteria.

Up to 40-50% of patients with aggressive B-cell non-Hodgkin lymphoma (B-NHL) remain refractory to treatment, or develop relapse (r/r) after 1-2 lines of therapy. The prognosis of this patient’s group remains unfavorable. However, the emergence of new methods of targeted drug and immunotherapy (polatuzumab vedotin, Pola; glofitamab) may improve both progression-free survival (PFS) and overall survival (OS) rates in the patients with r/r B-NHL. The aim of our study was to suggest the treatment strategy for this population of patients, as well as to determine place for allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Patients and methods

The study included 28 patients (pts) with r/r B-NHL treated with a bispecific drug (anti-CD20/anti-CD3) glofitamab (G) within Russian Early Access Program. G was administered at escalated schedule, i.e., 2.5 mg D8C1, 10 mg D15C1, 30 mg D1C2-12. Anti-CD20 antibody was administered 1 week before the G therapy was started. Overall response rates (ORR), progression-free survival (PFS), overall survival (OS) were estimated in the course of G therapy, with regard of prior treatment strategy. Treatment efficacy was assessed by PET-CT (Lugano criteria). Adverse events (AEs) were assessed by the NCI CTCAE 5.0.

Results

Median age at G initiation was 50 years (21-83); male/female ratio, 11/17 (39/61%). Median number of previous lines of therapy before G therapy was 3 (2-8). Autologous SCT was conducted in 7 pts (25%), polatuzumab vedotin (Pola) in 7 (25%) pts. ECOG status >1 before G initiation was registered in 7 cases (25%); B symptoms, in 6 pts (21%), and bulky disease was documented in 8 (29%) pts. Median follow-up was 6 (1-15.9) months. ORR was 67% in the total group: complete response (CR), in 15 (56%) pts; partial response (PR), in 3 cases (11%); stable disease (SD), in one patient (4%); disease progression (PD), in 8 cases (30%). Eight patients died during G therapy including 5 pts (18%) who deceased due to disease progression. Eight patients died at the time of analysis, including PD in 5 cases (18%). Median OS was not reached, 6-month OS was 75.1% (95% CI, 52.0–88.2); median PFS was 10.7 months (95% CI, 5.4-NA); 6-month PFS comprised 58.8% (95% CI, 35.9-75.9). Other factors, e.g., number of previous lines of therapy, r/r clinical course, auto-HSCT, Pola therapy did not influence both OS and PFS, and development of response to G. In the course of analysis, 22 (79%) pts discontinued therapy due to PD (n=10, 36%); 5 pts (18%) had severe COVID-19 infection; 5 pts (18%) completed the scheduled therapy, and 2 pts (7%) cancelled their treatment by other reasons. The median number of G cycles was 6 (2-12). COVID-19 of any grade was revealed in 9 (32%) pts. Three pts (11%) died due to severe COVID-19. The group of patients who received Pola-BR (n=7) before G therapy was also monitored: CR was achieved in 3 pts; PR, in 2 cases, and 2 pts developed PD. Among the patients who achieved CR during Pola-BR therapy (n=3), 2 pts had relapses during the therapy, and one patient, 11 months after Pola-BR completion. The achievement of response to Pola-BR did not influence clinical response to the G therapy.

Conclusion

New targeted and immunotherapeutic agents significantly improve clinical prognosis in the patients with r/r B-NHL. However, curative potential of such therapy has not yet been determined, thus requiring long-term observation, as well as selection of the patients who will benefit from allo-HSCT.

Keywords

Non-Hodgkin lymphoma, immunotherapy, targeted therapy, glofitamab, polatuzumab vedotin, adverse events.

Myeloid derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid derived cells, which includes early (E-MDSC; Lin^–HLA-DR^–CD33⁺), neutrophils and polymorphonucler myeloid-derived (PMN-MDSC; Lin^–HLA-DR^–CD66b⁺), and mature MDSCs (M-MDSC; CD14⁺HLA-DR^low/–), which, through various mechanisms of immunosuppression, are able to participate in the pathogenesis of multiple myeloma (MM) leading to tumor progression and antitumor therapy resistance mechanisms formation. We investigated the circulating MDSCs dynamics and arginase-1 suppressor molecule expression in patients with MM in the first 12 months after autologous hematopoietic stem cell transplantation (auto-HSCT) and the relationship between the MDSC content and auto-HSCT outcomes.

Materials and methods

The study included 44 patients with MM who underwent auto-HSCT. The counts of granulocytic (G-MDSCs; Lin^–HLA-DR^–CD33⁺CD66b⁺), monocytic (М-MDSCs; CD14⁺HLA-DR^low/–) and early MDSCs (Lin^–HLA-DR^–CD33⁺CD66b^–) were assessed by flow cytometry.

Results

After the patients underwent auto-HSCT, the relative amount of circulating M-MDSC (pU=0.038) and PMN-MDSC (pU=0.003) statistically significantly increased by the time engraftment. The absolute PMN-MDSC count also increased significantly (pU=0.005). Six months after auto-HSCT, the circulating М-MDSC and PMN-MDSC proportion significantly decreased (pU=0.007 and pU=0.024, respectively) to pre-transplant values and remained similar at 12 months after auto-HSCT. The relative and absolute number of E-MDSC at the time of engraftment, on the contrary, was reduced in comparison to similar values prior to auto- HSCT (for absolute values, it was statistically significant; pU=0.004). The circulating E-MDSC proportion among MNCs remained the same during the entire 12-month post-transplant period. While absolute number of MNCs increased, the absolute E-MDSC content also increased and was significantly higher 12 months after auto-HSCT compared to the period of engraftment (pU=0.032).

The analysis of relapse-free survival based on the median relative PMN-MDS value at engraftment (Me, 0.17%) showed that, in patients with PMN-MDSC counts of >0.17%, the proportion of patients free of early relapse was 94±6%. Meanwhile, in cases of PMN-MDSC ≤0.17%, only 67±11% patients were relapse-free, thus being significantly lower (p=0.049). In the group of patients with early recurrence, the proportion of M-MDSC expressing arginase -1 at the time of recovery from leukopenia showed a trend to lower values compared to the same index in the group of patients who maintained a response over the first 12 months of the post-transplant period.

Conclusion

The association of early MM recurrence after auto-HSCT with lower content of PMN-MDSC and reduced counts of arginase-1+ M-MDSC at the stage of recovery from leukopenia may suggest that MDSC is involved into restriction of homeostatic cell proliferation as a prerequisite for more effective immune reconstitution.

Keywords

Myeloid suppressors, autologous hematopoietic stem cell transplantation, multiple myeloma, suppressor molecules, arginase-1.

Chronic lymphocytic leukemia (CLL), as well as chronic kidney disease (CKD), mainly occur in the elderly (60-75 years) and senile (over 75 years) age. It is also known that a decrease in glomerular filtration rate (GFR) and the CKD presence have drastic influence on long-term prognosis. The aim of the study was analyzing the CKD occurrence in CLL patients and assessment of their impact on survival.

Materials and methods

A total of 132 CLL patients (60 men and 72 women) with known overall survival observed in GUZ UOKB hematology department from 01.2010 to 02.2020 were included in this retrospective cohort. Patients with HIV infection, tuberculosis and other paraneoplastic processes were excluded from the study. At the time of CLL diagnosis the GFR was calculated by CKD-EPI formula and CKD presence was assessed in accordance with CKD stage classification and stratification in accordance with GFR value. An end-to-end linear correlation analysis of the obtained Pearson indicators was carried out.

Results

The average patients’ age was 66.0±10.3 years. Among the studied cohort 64 (48.5%) patients had CKD with stage C2 found in 23 (17.4%), and stage C3a in 41 (31.1%) patients. This CKD rate in studied cohort significantly exceeds the one described by national epidemiological studies, which have detected CKD in 36% of studied patients older than 60 (Bikbov B.T. et al., 2009). The CKD structure depending on the CLL stage (Binet classification) was as follows: The CKD C2 stage was registered in 8.3%, 7.6%, and 1.5% of cases of CLL A, B, C, respectively. CKD C3a stage was diagnosed, respectively, in 9.8%, 18.9%, and 2.3% of CLL A, B, and C, respectively. It should be noted that there is no connection between CLL stage and CKD severity. However, this confirms the earlier published data stating no such relationship [Erten N. et al., 2005]. The studied cohort analysis showed only 43 (32.5%) patients to overcome estimated median survival rate in accordance with Binet classification. Of particular interest is the fact that a strong inverse correlation was found between C3a CKD and survival (p<0.05). We have previously found that initial GFR of more than 76.5 ml/min/1.73 m² in newly diagnosed CLL patient may predict the survival exceeding one calculated by median survival according to Binet classification (Patent RU No. 2725877 C1; 2020). Patients with CKD C3a do not belong to this category. In study conducted there were no patients with CKD C3a at the CLL diagnosis who did not exceed estimated median survival according to Binet classification.

Conclusions

In the cohort studied, the CKD C3a stage had a significant impact on CLL patients’ survival. Careful GFR monitoring starting at diagnosis will optimize the prognosis in this category of patients.

Keywords

Chronic lymphocytic leukemia, chronic kidney disease, overall survival.

Prognosis of patients with therapy-related myelodysplastic syndrome (t-MDS), developing after previous chemotherapy or radiotherapy therapy for primary hematological malignancies, is not well described. It is not clear if the prior history of hematological malignancy contributes to prognosis beyond the IPSS-R score. The aims of our research were to compare overall survival for patients with de novo MDS (d-MDS) and t-MDS after primary hematological malignancy and evaluate the role of allo-HSCT in prognosis with patients with t-MDS.

Patients and methods

A retrospective cohort single-center study included 94 patients, i.e., 23 cases of t-MDS with history of primary hematological malignancy, and 71 with d-MDS. OS was assessed with Kaplan-Meier curves, and differences between the groups were assessed using log-rank test. A multivariate Cox proportional hazard model was constructed to adjust for IPSSR-R prognostic group, age, and history of allo-HSCT.

Results

All patients were included into the analysis, with the median follow-up of 10 and 37 months for t-MDS and d-MDS, respectively. OS rate was significantly lower for t-MDS compared to d-MDS (p=0.04, log-rank test), with a median OS of 14 and 48 months, respectively. The treatment histories of primary hematological malignancy were associated with poor prognosis for patients, being independent on the IPSS-R and age (HR=1.9 [1.02; 3.37], p=0.04). At the same time, inclusion of allo-HSCT into the regression model made this factor not significant, with HR=1.2 [0.62; 2.27] (p=0.6). Allo-HSCT was performed in 30% of patients (n=7) with t-MDS and 73% of patients (n=52) with d-MDS. The frequency of transformation into acute myeloid leukemia (AML) and the median terms of AML transformation did not differ significantly for t-MDS and d-MDS groups (p=0.06 and p=0.27, respectively). Unfavorable cytogenetic risk, according to Armand risk score, was established in 20% (n=14) for d-MDS group and 70% (n=16) in the t-MDS group (p<0.001). The main characteristics of patients with t-MDS (primary diagnosis, stage of disease, durations of cytopenia, previous therapy) were also subject to analysis. Median time from cytopenia to the t-MDS diagnosis was 1.5 months. The structure of primary malignancies included classical Hodgkin’s lymphoma 48% (n=11), non-Hodgkin’s lymphomas 34% (n=8), acute leukemia 9% (n=2), and chronic lymphocytic leukemia 9% (n=2). The primary cancer status at the time of t-MDS diagnosis showed complete remission in 78% (n=18). Therapy for previous malignant neoplasms included chemotherapy alone in 44% (n=10) as well as combined chemotherapy and radiation treatment in 56% of cases (n=13). The predictive value of risk stratification proposed by MD Anderson Cancer Center Group for t-MDS (TPSS) was demonstrated (p=0.01). Regarding the OS rate among the t-MDS group was significantly higher for the patients who underwent allo-HSCT (p=0.02, log-rank test).

Conclusion

History of treatment of primary hematological malignancy can be independently associated with poor prognosis for the patients with t-MDS, being independent on the IPSS-R and age. However, uneven distribution of allo-HSCT frequency among the groups may probably be a confounding factor which leads to differential prognosis between these groups, thus requiring further research.

Keywords

Secondary myelodysplastic syndrome, therapy-related, allogeneic hematopoietic stem cell transplantation.

Blast crisis in chronic myeloid leukemia (CML BC) is a rare clinical and biologically heterogeneous condition often characterized by aggressive clinical course. Despite successful use of tyrosine kinase inhibitors (TKIs), some patients with CML still develop blast crisis. There is no standard approach to the BC treatment, and outcomes are, generally, unsatisfactory with long-term overall survival rarely exceeding 10% (Saxena K. et al.). The objective of this study was to evaluate treatment outcomes, in particular, overall survival (OS), in patients with CML BC.

Materials and methods

A total of 87 patients (65 males and 22 females) with verified CML treated at the RM Gorbacheva Research Institute for 1st or subsequent BC were included into this retrospective cohort. Therapeutic efficacy was evaluated according to the European LeukemiaNet (ELN) criteria.

Results

The median age at the time of diagnosis was 42 (18-62) years. In 66% of cases (n=57), the diagnosis was established in chronic phase (CP); in 19% of cases (n=17), in acceleration phase (AP). De novo occurrence of BC was revealed in 15% of cases (n=13) identified as myeloid in 9%, and lymphoid, in 6% of the patients. Cytogenetic data at diagnosis were available in 85 cases (98%). Additional chromosomal aberrations (ACAs) were present in 31 (37%) cases. The most frequent findings were, e.g., trisomy 8 (29%), 3q26 rearrangement (23%), monosomy 7 / 7q deletion (17%), trisomy 19 (13%), additional Ph-chromosome (13%), and complex karyotype (55%).

The median age at the first BC was 44.5 (21-76) years, with a median time since primary diagnosis of 39.5 (0-248.5) months. Most BC cases (79.3%) were myeloid; extramedullary lesions were seen in 8% of cases. Most patients received complex therapy regimens combining chemotherapy and TKIs (56%); only chemotherapy was applied in 30% cases, and in 14% cases TKI-only regimens were used, mostly, with dasatinib (in 75% of cases). Allogeneic hematopoietic stem cell transplantation was not performed due to progression, severity of condition, presence of significant comorbidities, lack of donors, patient refusal. The response was evaluated in 78% of cases, with 47% of patients being resistant to 1^st-line therapy and 31% of patients reaching CP (complete hematologic response in 100%, cytogenetic response in 30%, and molecular response in 19% of cases). In 22 % of cases, a 2^nd BC developed, with median OS of 11.5 months since 1^st BC. The median time from BC1 to BC2 or death evaluated in 72 patients was 7.8 (0.6-107.2) months. At the time of the last evaluation, 16% of patients were alive and 84% died of the disease progression (82%), other documented reasons (1.4%), or due to unknown cause (17.4%). The 2-year and 5-year OS were 28.7% and 18.4%, accordingly.

Conclusion

The outcomes in patients with CML BC remain poor, with a median time until BC2 development or death being less than 1 year. Prompt diagnosis and treatment initiation as well as timely transition to allogeneic hematopoietic stem cell transplantation are crucial to achieve a response.

Keywords

Chronic myeloid leukemia, blast crisis, tyrosine kinase inhibitors.

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent immunotherapeutic. NK cells’ mature phenotype and abundance positively correlate with a favorable prognosis in acute myeloid leukemia (AML) and prolonged treatment-free survival in chronic myeloid leukemia (CML). However, NK cell maturation and cytotoxicity are suppressed during the disease, and the environmental drivers of this impairment are not fully characterized. Given the established role of inflammatory cytokines (e.g., IL 1β, IL-6, TNFα) in the progression of myeloid malignancies, we hypothesize that leukemic inflammation impedes NK cell cytotoxicity shifting them toward a pro-tumor phenotype.

Materials and methods

To define the effect of leukemic cytokines on NK cells, we utilize BCR-ABL⁺ (CML) and Flt3-ITD/TET2^−/− (AML) chimeric mice, as well as peripheral blood and bone marrow mononuclear cells from AML and CML patients. We establish the mouse models by transplanting CD45.2⁺ C57BL/6 bone marrow cells carrying leukemic mutation(s) into CD45.1⁺ healthy hosts. Such chimeras represent a robust pre-clinical tool to study non-transformed immune cells during leukemia. Here, we profile CML- and AML-exposed NK cells using flow cytometry and single-cell RNA-sequencing (scRNA-seq).

Results

Consistent with clinical observations, NK cells are reduced in AML and CML mice, display an immature phenotype, and decrease surface activating receptors while upregulating inhibitory molecules Lag-3, TIGIT, and NKG2A. Moreover, leukemia-exposed NK cells show impaired cytotoxicity measured by target-specific degranulation ex vivo. Next, we mapped the transcriptional landscape of NK cells from control and CML mice. The scRNA-seq confirms reduced expression of NK maturation and cytotoxicity markers (Itgam, Cx3cr1, Prf1, Gzma) in leukemia. Among gene sets enriched in CML-exposed NK cells were those associated with cell division and inflammatory cytokine response; the latter include genes for cytokines (IL 1β, TNFα, GM-CSF), cytokine receptors, and negative regulators of STAT3/5 signaling. Pathway enrichment analysis revealed activation of IL-6/STAT3 and NF-kB signaling cascades – an effect likely triggered by inflammation. Thus, we next tested the impact of the leukemic environment on healthy NK cells. We found that serum from CML mice dampens NK cell degranulation ex vivo. Further ELISA identified elevated IL-6, IL-1α/β, TNFα, and GM-CSF in CML serum. Many of these cytokines can be secreted by functionally skewed NK cells, as indicated by scRNA-seq. RT-qPCR on sorted NK cells confirmed increased IL-1β, TNFα, and GM-CSF mRNA levels in CML suggesting NK cell secretory phenotype exacerbating leukemic inflammation. To validate the clinical relevance of our findings, we finally characterized NK cells from leukemic patients and healthy donors. In agreement with published data, NK cell frequencies are reduced in our cohort, along with altered expression of NK activating (NKG2D, DNAM-1) and inhibitory (KIR2DL1, NKG2A) receptors and diminished K562-dependent degranulation. Mirroring the mouse scRNA-seq data, patient NK cells possess a pro-inflammatory gene signature with the activation of TNFα, NF-kB, and PI3K-Akt signaling. Thus, NK cells are sensitive to disease-associated inflammation that interferes with their anti-leukemic cytotoxicity.

Conclusions

We speculate that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing an optimal target for NK-boosting immunotherapies.

Keywords

NK cells, inflammation, acute myeloid leukemia, chronic myeloid leukemia, immunotherapy.

Life expectancy of the patients with low- and intermediate-risk myelodysplastic syndrome (MDS) does not always correspond to the IPSS prognostic system (IPSS-R). According to the literature data, deterioration of clinical prognosis is associated with distinct factors, e.g., lack of response to therapy, infectious complications due to neutropenia, iron overload caused by blood transfusions, presence of the bone marrow fibrosis, development of secondary MDS after previous chemo- and radiotherapy. The predictors of potential transformation into refractory anemia 1-2 with blast excess and acute myeloid leukemia are insufficiently studied so far. Our study was aimed for determining the predictors of unfavorable course of myelodysplastic syndrome in the patients with low-to-intermediate risk of IPSS-R.

Materials and methods

We observed a cohort of 136 patients classified into the very low/low/intermediate risk group according to IPSS-R. The median age at the time of diagnosis was 49.5 years (18-76), with median follow-up period of 1019 days (42-9740). The male-to-female ratio was 62:74. A P-value of less than 0.05 was taken as statistically significant.

Results and discussion

Five-year overall survival was 59.5% (95% CI: 47.0-70.0, median not reached); 5-year progression-free survival was 44.6% (95% CI: 32.9-55.6, median 49.8 months). The five-year cumulative progression incidence was 40.7% (95% CI: 29.5-51.5), mortality without progression was 15.6% (95% CI: 8.4-24.8). Based on the results of univariate analysis of the five-year overall survival, the factors with a p-value of less than 0.15 were selected: age at initial diagnosis, presence of bone marrow stromal fibrosis, serum ferritin level at the time of diagnosis. These factors were further included into the proportional risks model. According to the results of multivariate analysis, only age at the time of diagnosis provided a statistically significant effect on five-year overall survival (p<0.03).

Conclusion

Comparable overall and non-progressive survival rates suggest a predominant effect of progression-free mortality on the clinical outcomes upon therapy in the patients with low-risk MDS. The data obtained indicate the heterogeneity of the patient group with very low/low/intermediate risk MDS, along with absence of convincing clinical predictors of progression. It is necessary to search for additional molecular biology prognostic factors in order to identify the patients with low- and intermediate-risk MDS with higher probability of disease progression.

Keywords

Myelodysplastic syndrome, clinical outcomes, prognostic factors.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently considered the golden standard treatment for young patients with aplastic anemia. Recovery of fertility (FR) after allo-HSCT has a significant influence upon the quality of life in the transplant survivors. The aim of the study was to evaluate the impact of conditioning regimen on fertility recovery in AA patients.

Patients and methods

A total of 62 patients with AA who survived for, at least, 1 year following allo-HSCT was included in our study. Most patients received busulfan-based (Bu) conditioning regimen, i.e., 39 patients (64%, group 2), and other 22 patients (36%) received cyclophosphamide-based (Cy) regimen (group 1). The median follow-up period was 5.5 (0.82-20) years. Most patients in group 1 were males (n=13, 57%). Nineteen (82%) patients received allo-HSCT from matched related donor (MRD); 4 (17%), from matched unrelated donor (MUD). The median age at allo-HSCT was 25 (17-38) years. Severe chronic graft-versus-host disease (GVHD) was registered in 2 cases (10%). Most patients in group 2 were females (n=21, 55%). Twenty-one patient (54%) received allo-HSCT from MUD, 18 (46%) were transplanted from MRD. Median age at allo-HSCT was 28 (17-50) years. Severe GVHD cases were registered in 3 cases (8%).

Results

Twenty-five patients (41%) had children before allo-HSCT. Twenty-three patients (38 %) did not plan to have children in the near future. 7% of patients underwent hysterectomy before allo-HSCT. Recovery of the menstrual cycle after allo-HSCT has been reported in 46% of cases (14 patients). Median time period from allo-HSCT to the menstrual cycle recovery was 2.3 (0.24-4.48) months. In 17 patients (54%), the cycle was not restored. Among the females who underwent allo-HSCT, nine pregnancies were registered which resulted in healthy live births. In the Bu-based group, one spontaneous pregnancy was registered, and one patient underwent IVF with a donor egg (3%). The men from the first group (Cy-based conditioning) had 5 children after allo-HSCT, and 6 children were born of them in second group (Bu-based conditioning). One patient from group 2 had to use IVF twice. А total of 21 pregnancies were registered. When analysing possible risk factors, only busulfan-based conditioning was a significant risk factor for impaired FR (p<0.03) and menstrual cycle recovery (p<0.03). Neither age of patient, type of donor, nor chronic GVHD had significant impact on fertility.

Conclusion

In our study, usage of busulfan in the conditioning regimen had a statistically significant impact upon recovery of fertility and the menstrual cycle after allo-HSCT.

Keywords

Aplastic anemia, hematopoietic stem cell transplantation, conditioning regimen, fertility.

Aplastic anemia (AA) is a rare severe blood disease characterized by pancytopenia and aplasia of bone marrow due to profound deficiency and functional defects of hematopoietic stem cells. In Kyrgyzstan, immunosuppressive therapy (IST) with equine antithymocytic globulin (ATGAM)+ Cyclosporine A is used to treat aplastic anemia since 2010. The incidence of AA in Asia is 2-3 times higher than in Western countries, where it is 1-2 cases per 1 million population per year. Our purpose was to assess results of long-term treatment in the patients over 60 years old with severe aplastic anemia against IST (ATGAM+Cyclosporine A).

Materials and methods

18 patients with severe aplastic anemia (SAA). Of these, there were 11 males and 7 females. The median age was 64 years (60-74 years). IST treatment protocol (ATG+Cyclosporine A) was as follows: 20 mg ATG per 1 kg body weight for 1-5 days, 10 mg Cyclosporine A\kg body weight on day 20. The median follow-up time for patients was 36 (3-72) months.

Results

Clinical and hematologic improvement after the first course of IST was observed in 13 patients (72.2%), response was absent in the remaining 5 (27.8%) patients, including one patient with extra-severe AA. None of the patients had complete or partial remission. After 12 months of the first course of IST, complete remission occurred in 3 (16.6%) patients, partial remission in 5 (27.7%) patients, clinical and hematological improvement was observed in 5 (27.7%) patients. In four patients (22.2%) out of 5 among those where there was no effect after the first course, the situation did not change. Event-free survival within the next 12 months was observed in 13 patients (72%).

Conclusion

Pathogenetic therapy of ATG + Cyclosporine A in patients over 60 years old has been implemented in Kyrgyzstan. It has shown good results being recommended for usage in AA treatment protocols in the Kyrgyz Republic.

Keywords

Aplastic anemia, elderly, immunosuppressive therapy, antithymocytic globulin, cyclosporine A.

Graft failure is among the key problems of allogeneic hematopoietic stem cell transplantation (allo-HSCT). This issue is extremely important in case of reduced-intensity conditioning regimens, when a great number of host T-cells still retains in the body. The evolutionary divergence of HLA seems to be interesting, due to its translation to the peptide diversity (including donor ones) which can be presented to residual host T cells. The Grantham distance is a measure for the evolutionary divergence, with its higher levels reflecting the larger number of peptides potentially presented to T-cells via the HLA system. Our aim was to evaluate the impact of immunopeptidome diversity upon development of graft failure in the patients with acute leukemia.

Patients and methods

The present study included 118 patients with acute leukemia, who underwent allo-HSCT at the National Research Center for Hematology (Moscow, Russia). We calculated the Grantham distance for the donor class I HLA determined by the high-resolution HLA-typing using a next-generation sequencing platform. Then we evaluated effects of the Grantham distance on the probability of the post-transplant graft failure. ROC analysis showed that the Grantham distance of >7.4 for HLA-A was associated with higher risk of graft failure. A multivariate analysis was carried out using Cox proportional hazards model, to assess the influence of different factors upon the risks of graft failure. The model included all known factors potentially affecting the graft failure (gender, age, type of conditioning, type of donor, graft source) as well as the Grantham distances for HLA-A.

Results

When analyzing possible effects of different demographic and transplantation-associated factors upon clinical course of the post-transplant period, we have shown that the Grantham distance >7.4 for the donor HLA-A, along with the type of donor, was associated with increased risks of the graft failure (HR, 5; p=0.043).

Conclusions

One may presume that higher diversity of the donor peptide numbers presented to residual host T cells may cause increased risks of immune response against donor cells, thus potentially leading to development of graft failure.

Keywords

Graft failure, allogeneic hematopoietic stem cell transplantation, HLA system.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment option for many children hereditary diseases of the immune or hematopoietic system, as well as ones with malignant neoplasms. The organization and conduct of HSCT are difficult and costly and not all countries have the resources and trained personnel to establish a HSCT program. Nevertheless, in 2016, a HSCT department with 7 beds was opened at the Hematology Center named after Prof. R. Yeolyan (HC), where auto-HSCT was performed up until 2021. A total of 75 auto-HSCTs were performed, 19 of which were in children. In April 2021, an allogeneic HSCT project was launched at the HC in the framework of cooperation with the Ministry of Health, the City of Smile Foundation (Armenia), DKMS (Germany) and Cure2Children (Italy). The main goal of the project is to develop allogeneic HSCT in Armenia and offer free or highly subsidized HSCT to African children with Sickle cell anemia (SCA) under the supervision of international HSCT experts.

Materials and methods

SCA with frequent crises and severe thalassemia is curable with HSCT resulting in normalization of hematopoiesis and disease symptoms attenuation resulting in quality of life improvement. The first two patients who underwent HSCT at HC were SCA patients with HLA-matched related donors. Prior to starting the conditioning regimen and placing a central catheter, exchange transfusions were performed in order to reduce HbS to <30%. Also, 10 ml/kg of the patient’s blood was removed within 1 hour, followed by an exchange transfusion at the rate of 15 ml/kg within the next three hours. Myeloablative conditioning regimen including Fludarabine 30 mg/m²/day from day -18 to -13 (total 180 mg/m²), ATG (Rabbit) at total dose of 4 mg/kg on day -12 to -10, Busulfan 14 mg/kg given on day -9 to -6, Cyclophosphamide 200 mg/kg/day from day -5 to -2 followed by the infusion of freshly harvested HLA-compatible G-CSF-primed marrow on day 0. Graft-versus-host disease (GVHD) prophylaxis consists of Cyclosporine A and Methotrexate. Chimerism is evaluated on D+30, D+60, and D+ 90. In order to evaluate the results of transplantation, hemoglobin electrophoresis is also performed.

Results

The patient is a 9 years old girl from Nigeria with SCA who was admitted to the HC in September 2021 with complaints of frequent abdominal pain, mucosal icterus. The patient’s donor was a match related sibling. The child received hydroxyurea prior to admission to the HC at a dose of 20-30 mg/kg/day, considering hepatosplenomegaly. During the examination in the HC, before HSCT, was diagnosed with bilirubinemia (total bilirubin 60.0-80.0 µmol/l, indirect bilirubin 54.0-70.0 µmol/l). A blood test was conducted at the Genetic Center and a diagnosis of Gilbert’s syndrome was made. After consultation with experts it was decided to perform HSCT according to the protocol without reducing drug doses or changing conditioning regimen. The transplantation proceeded without complications. Engraftment was on D +20. Bilirubin began to decrease immediately after D0. The patient developed grade 1 aGVHD of the skin and GIT, which resolved without further treatment. Currently, the child’s condition is stable; the dose of Cyclosporine is tapered.

Conclusions

This case illustrates our experience of allo-HSCT performed in a patient with SSA.

Keywords

Sickle-cell anemia, thalassemia, children, allo-HSCT, Armenia.

The standard of treatment for pediatric patients with severe acquired aplastic anemia (SAA), who do not have an HLA-compatible related donor to perform allogeneic hematopoietic stem cell transplantation (HSCТ), is the combined immunosuppressive therapy (IST). An unsatisfactory result of IST motivates to search for factors that determine the need for allo-HSCT from an unrelated HLA-identical donor as first-line therapy. This retrospective study was designed in order to search for early unrelated donor allogeneic HSCT decision-making criteria in children with SAA.

Materials and methods

Full exome sequencing was carried out on archival initial BM samples of 58 patients younger than 18 years with SAA treated at the Research Center for Pediatric Oncology, Hematology and Immunology in 1997-2018.

Results

Somatic mutations in 43 genes were detected in 32 out of 58 (55.2%) children with SAA. They formed a determined molecular genetic status (DMGS) group. Patients without DNA mutations were assigned to unknown molecular genetic status (UMGS) group. Only 46 patients receiving IST were included in the analysis aimed to determine genetic mutations of clinical significance in SAA. The overall survival rate for patients with mutations at the time of diagnosis and after immunosuppressive therapy is lower than for patients without mutations. (75%±9% and 81%±8%, respectively). All patients with mutations were divided into 2 subgroups based on mutations identified. The overall survival of patients with mutations in genes associated with immunological dysregulation (IL21R, CASP10, LYST, FAS, ADA2, XIAP) was lower (60%±22%) than in patients with other molecular changes (80%±9%) and patients without mutations (81%±8%). The overall survival of patients with mutations causing impaired DNA repair (FANCB, FANCC, SOS1, NBN, RUNX1, ACD, TERT, POLD1, MSH3, BRCA2, POLE, ATR, BRCA1, MSH2, RAD54L, MSH6, MLH1, MLH3, ATM, NABP2, NEIL1) was also lower (71%±11%) than in patients with other molecular genetic changes (86%±13%) and patients without mutations (81%±8%). The overall survival of patients with a single mutation was significantly lower (56%±17%) compared to patients with multiple mutations (86%±12%). The overall survival of patients with a mutation in the MSH3 gene was 0%. The overall survival of patients with mutations in the POLE+POLD1 genes was 33%±27% and was significantly lower (p=0.0093) than in patients without these disorders (81%±9%). All patients with mutations in the MSH3 and POLE+POLD1 genes were combined into a high risk group characterized by worse response to IST. The overall survival of patients in this group was 33±19% and was significantly (p=0.0029) lower than in patients without mutations in these genes (89±7%). It was found that when the number of colony forming units of granulocytes and monocytes (CFU-GM) is below 2.5×10⁵ and the number of burst-forming units of the erythroid series (BFU-E) is below 0.1×10⁵ with growth factors it is necessary to exclude the presence of mutations in the MSH3, POLE and POLD genes (sensitivity 100%).

Conclusions

The presence of mutations in the MSH3, POLE+POLD1 genes is an unfavorable predictor for long-term overall survival in patients with SAA. These patient require allo-HSCT from unrelated HLA-identical donor as first-line therapy.

Keywords

Severe aplastic anemia, stratification, molecular genetics, allo-HSCT.

TCRαβ+/CD19+ cells depletion allows reducing the risks of severe complications after haplo-HSCT and also retains NK-cells in the graft. In this retrospective analysis we attempt to focus on the relationship between simple NK-related factors, namely KIR mismatch, NK graft content, peripheral blood NK cells counts on day +30 after HSCT and transplant outcomes, such as relapse or transplant-related mortality (TRM) incidence, in a cohort of children with acute leukemia transplanted in complete remission.

Materials and methods

The study cohort includes 296 patients diagnosed with ALL (acute lymphoblastic leukemia) in 190, AML (acute myeloblastic leukemia) in 94, and ABL (acute biphenotypic leukemia) in 12 cases, accordingly. All patients received their first haplo-HSCT with αβ T cell depletion from January 2012 to April 2021. KIR match or mismatch was predicted based on ligand-ligand model for all patients. Patients cohorts were divided by median absolute count of graft NK cells subpopulation and peripheral blood NK cells count on day +30 after the HSCT. The relapse and TRM risks were calculated for each group by cumulative risk method, groups were compared by Gray test.

Results

The cumulative relapse incidence was 28.6% (23.5%-34.9%) in the whole cohort, 28.6% (22.6%-36.3%) among patients with ALL, 26.8% (18.3%-39.4%) among patients with AML, and 42.7% (19.6%-93.2%) in the small ABL cohort. There were 16 non-relapse mortality cases in the whole cohort. The cumulative TRM incidence was 5.9% (3.7%-9.6%) in the whole cohort, 8.1% (4.9%-13.5%) for ALL, and 2.1% (0.5%-8.4%) for AML patients. KIR mismatch was predicted for 32.7% donor-recipient pairs. We failed to detect a correlation of KIR mismatch and relapse or TRM incidence either in whole cohort or in smaller ALL and AML cohorts. There were only 2 TRM cases in the AML group, both of them inside KIR-mismatched cohort. No correlation was detected between NK cells dose in the graft and leukemia relapse incidence in the whole cohort, while there was a trend for lower relapse rate in ALL patients receiving higher NK-cells dose (p=0.140). There was also a trend to higher TRM in patients with receiving higher than median NK-cells dose (p=0.135 for total cohort, p=0.087 for ALL cohort). The higher dose of NK cells has a trend to correlate with lower leukemia relapse risk in the group with donor-recipient KIR mismatch (p=0.06) and there was no correlation in the KIR-matched patients’ cohort. Among patients with AML without KIR mismatch, surprisingly, higher relapse incidence was seen among patients with higher dose of NK cells in the graft (p=0.002). In the whole AML patients’ cohort higher peripheral blood NK cells counts on day +30 also correlated with higher relapse risk (p=0.025).

Conclusions

A higher dose of NK cells in the graft was associated with a lower relapse risk for KIR mismatch patients with acute leukemia, both ALL and AML. Paradoxically, higher dose of graft NK cells was associated with higher relapse risks in patients with AML receiving KIR-matched HaploHSCT.

Keywords

Hematopoietic stem cell transplantation, TCRαβ+/CD19+ depletion, acute leukemia, immune reconstitution, HSCT complications.

The REACH III trial completed in May 2020 has shown ruxolitinib to be the best graft-versus-host disease (GVHD) therapy option currently available. Still, up to 30% of patients with GVHD are refractory to this therapy and are left without standard-of-care options.

Materials and methods

We conducted a retrospective, single-centered study in a cohort of patients refractory to ruxolitinib as monotherapy. A total of 26 patients were then treated by ruxolitinib in combination with extracorporeal photopheresis. The median age was 20 (3-46) years with 57.7% (n=15) of patients being adults and 42.3% (n=11) being children. M/F ratio was 13/13. Most patients with acute GVHD had Gr 3 (76.9%, n=20) or Gr 4 (7.7%, n=2) disease at therapy initiation. Also, 61.5% (n=16) of patients had moderate to severe chronic GVHD. In 92.3% (n=24) there was skin, in 73.1% mucosa, in 61.5% eyes, in 53.8% liver, and in 23% joints involvement.

Results

During the treatment, 61.5% (n=16) patients achieved a partial response. The overall survival (OS) rate was 92.3% (n=24), progression-free survival was 88.5% (n=23). In 19.2% of patients (n=5) immunosuppressive therapy (IST) was successfully withdrawn. The median time to partial response (PR) was 42 (4-730) days and median therapy duration was 61 (7-1095) days. The best response rate was seen in patients with GVHD of the mucosa (30.8%, n=8). Skin and liver GVHD had the same response rate of 23.1% (n=6 for each). Patients with gastrointestinal tract (GIT) GVHD had a response rate of 26.9% (n=7). Patients with joint and genital GVHD had the same response rate of 11.5% (n=3). Only 15.4% (n=4) responses were seen in lung GVHD cases. Patients with ocular GVHD had shown disease progression in 19.2% (n=5). The patients with skin, joint, mucous membranes, and gastrointestinal tract GVHD, exhibited the disease progression in 7.7% of cases (n=2), accordingly. In patients with genital and pulmonary GVHD, disease progression was observed in only 3.8% cases (1 person). The vast majority of patients had no complications. In 11.5% (n=3) patients there was CMV reactivation, 7.7% of patients (n=2) developed hemorrhagic cystitis.

Conclusions

In total, 61.5% of patients respond to combined therapy with ruxolitinib and extracorporeal photopheresis. The vast majority of cases the symptoms show no signs of worsening. Thus, ruxolitinib combined with extracorporeal photopheresis seems to be a feasible therapy approach in patients with resistant GVHD. Due to the small sample size, validation on larger patient groups is necessary.

Keywords

Graft-versus-host disease, ruxolitinb, extracorporeal photopheresis, steroid resistance.

Acute and chronic steroid-refractory “graft-versus-host disease” (srGVHD) is one of the most severe complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT), indicating the inefficiency of glucocorticosteroids, which are the standard of the first line therapy. There are no standards of 2^nd-line therapy for srGVHD. There is evidence of the successful use of JAK-inhibitors as a treatment for GVHD in adults and in children over 12 y.o. There are no data of the use the Ruxolitinib in the pediatric cohort up to 12 y.o. Our objective was to evaluate the efficacy and safety of ruxolitinib in the treatment of srGVHD after allo-HSCT in children.

Patients and methods

The study included 68 patients (49 boys and 19 girls) with srGVHD who received ruxolitinib in the second or subsequent lines of therapy. The median age – 5.5 y.o. (1-18). The largest number of patients were diagnosed with acute leukemia (ALL – 25, AML – 14, JMML – 5), MPS I type – 6, aplastic anemia – 5, and 7 patients had non-malignant diseases. Allo-HSCT from match related donor was performed in 3 patients, match unrelated donor – 22, haploidentical – 43. Myeloablative and non-myeloablative conditioning regimens were performed in an equal number of patients. All patients received cyclophosphamide-containing GVHD prophylaxis. Thirty-seven patients had acute GVHD (aGVHD), 31 – chronic GVHD (cGVHD). At the time of inclusion in the study, 10 patients had grade II aGVHD, 15 – grade III, and 12 grade IV aGVHD. Skin lesions 2-4 gr. observed in 89%, gastrointestinal tract 2-4 gr. – 48%, liver 3-4 gr. – 8%. Eight patients had moderate cGVHD and 22 – severe. The most frequently was the skin lesion of 2-3 (87%), mucous membranes of 2-3 (41%) and eye damage of mild severity (48%). The median dose of ruxolitinib was 0.3 mg/kg for aGVHD and 0.25 mg/kg for cGVHD. Median intake was 122 days for aGVHD, 341 days for ccGVHD.

Results

Complete response rate in patients with aGVHD was 64%, partial – 18%. The complete response rate for cGVHD was 35%, partial – 51%. No response or disease progression occurs in 15% with aGVHD and 12% with cGVHD. Median time to overall response was 32 days (6-110) for aGVHD and 50 days (7-496) for ccGVHD. The most common complication was the development of hematological toxicity in the form of thrombocytopenia 3-4 degrees (50%) and neutropenia 3-4 degrees (59%). Long-term use of IST led to an isolated increase in liver transaminases of 3-4 degrees was in 33% of patients. Due to the long-term use of immunosuppressive therapy 63% of patients with aGVHD and 54% of patients with cGVHD had viral infections, which required systemic antiviral therapy. The overall survival of patients with aGVHD was 59%, with cGVHD – 86% (p=0.07). The severity of GVHD, the time to achieve an overall response, the type of allo-HSCT didn’t significantly affect the overall survival in both acute and cGVHD (p>0.1). Also, there was no effect on the overall survival of the fact of the addition of bacterial, viral or fungal infections during therapy with Ruxolitinib.

Conclusion

Ruxolitinib showed good efficacy with relatively low toxicity and can be used as an option for the treatment of acute and chronic GVHD as a 2^nd or subsequent line of therapy in a pediatric cohort of patients.

Keywords

Graft-versus-host disease, steroid-refractory, ruxolitinib, hematopoietic stem cell transplantation.

Chronic GVHD (cGVHD) is one of the most common late complications of allo-HSCT that affects relapse-free survival rates and quality of life in the patients. T-helper cells (Th) play one of the key roles in the pathogenesis of cGVHD. Our objective was to compare the amounts of Th cell subpopulations in peripheral blood (PB) of the patients with cGVHD and those without this complication at +180 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Materials and methods

49 patients who underwent allo-HSCT for acute leukemia (27 AML, 22 ALL) were included into the study. Their median age is 40 years (20 to 58). Allo-HSCT was performed from related haploidentical donor in 32 patients (65%), the rest, others received grafts from compatible unrelated donors. cGVHD was developed in 18 (37%) patients. PB samples were taken at the day +180 after allo-HSCT and were analyzed by multicolor flow cytometry by means of “CytoFLEX” device (Beckman Coulter). As based on expression of CD25, CD3, CD4, CD8, CXCR3, CCR6, CCR4, CCR10, CXCR3, CXCR5, the following cell populations have been identified: Th type 1 (Th1), Th type 2, Th type 17, Th22 type (Th22), T-follicular helpers, T-regulatory cells. When measuring expression of CD45RA, CCR7 and CD45RA, these populations were discerned according to their degree of maturation, e.g., naive cells, central memory (CM) cells, transient memory, effector memory (EM) and terminally differentiated cells.

Results

On day +180 after allo-HSCT, blood samples from the patients suffering from cGVHD contained significantly higher absolute counts of Th cells (in cGVHD cases, 104 cells/μl; in cGVHD-free patients, 62 cells/μl, p=0.005), as well as Th1 CM-phenotype (in cGVHD patients, 1.91 cells/μl; without cGVHD, 0.88 cells/μl, p=0.021), and Tx22 CM cell phenotype (in cGVHD, 1.03 cells/μl; without cGVHD, 0.44 cells/μl, p=0.042). However, we revealed lower absolute counts of Th EM phenotype (cGVHD, 6 cells/μl; cGVHD-free, 16 cells/μl, p=0.023), and of Th1 EM phenotype (in cases of cGVHD, 1 cell/μl; without cGVHD, 6 cells/μl, p=0.004).

Conclusion

The role of both Th1 and Th22 cells in pathogenesis of cGVHD has been highlighted. IFNy is the main product of Th1 population. IFNy induces the synthesis of CXCL9, CXCL10 and CXCL11 chemokines which are recognized as plasma markers of cGVHD, i.e., their levels are increased at the time of diagnosis and remain high throughout active phase of the disorder. The CXCL9 level also correlates with cGVHD severity and shows a prognostic potential. Interleukin-22 (IL-22) is the main product of Th22 activity. The IL-22 contents correlate with severity of skin lesions in cGVHD. Some “shift” of the phenotype to the central memory pattern may be associated with both damage to the thymus and the applied immunosuppressive therapy. This question seems to us extremely interesting and requires further study in larger samples.

Keywords

T-helpers, allo-HSCT, chronic GVHD.

Efficacy of salvage allogeneic hematopoietic stem cell transplantation (HSCT) in myeloid neoplasms not responding to chemotherapy and targeted drugs remains limited. Our group have recently demonstrated augmented graft-versus-leukemia (GVL) effect with substituting cyclophosphamide with bendamustine in graft-versus-host disease (GVHD) prophylaxis regimen (Moiseev et al., TCT, 2021). Nonetheless, this original regimen was associated with significant toxicity due to poorly controlled cytokine release syndrome (CRS). To overcome this limitation we conducted a pilot single-center study of GVHD prophylaxis with a combination of cyclophosphamide with bendamustine in refractory myeloid malignancies.

Materials and methods

The prospective (NCT04943757) Phase I single-arm study evaluated GVHD prophylaxis regimen consisting of bendamustine 50 mg/m²/day on days +3,+4, cyclophosphamide 25 mg/kg/day on days +3,+4 (PTCBCy), tacrolimus 0.03 mg/kg from day+5 to day+100 and mycophenolate mofetil 30 mg/kg/day on days 5-35. Patients received reduced intensity FB2 or FB3 conditioning according to performance status. Main inclusion criteria were: diagnosis of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) or blast crisis of chronic myeloid leukemia (CML) or other myloid neoplasms (MPN) with high tumor burden, no hematological response to previous therapies, absence of severe organ dysfunction. Thirty patients were included into the interim analysis. The median age was 42 years (range 18-69). AML was an indication for HSCT in 22; MDS, in 6, and 2 patients had CML and MPN.

Results

Median follow-up at the time of the analysis was 5 months (range 2-18). Engraftment was documented in 87% of patients. Median time to engraftment was 18 days (range 12-35). Complete response (CR) was achieved in 83% of patients and 73% were minimal residual disease (MRD)-negative. With the limited follow-up, overall survival was 67% (95%CI 43-82%), while event-free survival (including graft failure as event) was 36% (95%CI 14-58%). Disease progression or relapse was the major cause of failure and was documented in 55% of patients (95%CI 26-76%). On the other hand, the combination regimen was associated with low toxicity and GVHD incidence. The cumulative incidence of Grade II-IV acute GVHD was 3% and observed in one patient. Additionally, 5 patients had grade 1 acute GVHD. In landmark analysis at 100 days, the incidence of chronic GVHD was 24% (95%CI 5-50%). Chronic GVHD manifested as liver overlap syndrome in the majority of these patients. CRS was documented in 20% of patients, with grade 4-5 in only 2 cases. Most common target of CRS was liver (17%), pancreas (10%) and kidneys (7%). CRS was effectively controlled by tocilizumab, ruxolitinib and high dose steroids in all cases, except of one patient. All CRS cases were associated with increased serum ferritin (median, 26400 ng/ml; range 12570-206500). Non-relapse mortality was 7% (95%CI 1-19%). Preliminary flow cytometry analysis demonstrated the same pattern of early immunological recovery, preservation of central memory T-cells and induction of tolerance by PD-1L positive monocytes as in the single-agent bendamustine study.

Conclusions

This pilot trial demonstrated that PTBCy combination prophylaxis provides the level of safety compared to conventional GVHD prophylaxis regimens with maintenance of GVL patterns. The study continues enrollment of patients.

Disclosure

The authors declare no conflicts of interest.

Keywords

Myeloid malignancies, allo-HSCT, graft-versus-host disease, posttransplant cyclophosphamide, posttransplant bendamustine.

Sepsis is a life-threatening clinical condition in immunocompromised patients, especially after hematopoietic stem cell transplantation (HSCT). Sepsis could lead to unregulated inflammation, causing multiple organ failure and hypercytokinemia with lethal outcome. Urgent hemosorbtion could be considered a potential option in complex therapy of sepsis after HSCT. Our aim was to estimate effectiveness of hemoperfusion with polymyxin B-immobilized cartridge (PMX-hemoperfusion) in the early posttransplant period.

Patients and methods

PMX-perfusion was performed in 3 patients who developed Gram-negative sepsis at early terms after HSCT. Pt. 1: Female, 15 y.o., ALL relapse, HSCT from MUD, conditioning: TBI/VP/Flu/ATG, +15 d. after HSCT as the start day of PMX-hemoperfusion. WBC: 1000/mcL. Pt. 2: Female, 15 y.o., AML relapse, HSCT from haploidentical donor with TCR a/b/CD19-depletion. Conditioning: FLAM followed by Treo/Mel. Day at the start of PMX-hemoperfusion: +20. WBC: 2500/mcL. Pt. 3: Male, 17 y.o., ALL relapse, HSCT from haploidentical donor with TCR a/b/CD19-depletion. Conditioning: TBI/VP/Flu. Day at the start of PMX-hemoperfusion: +20. WBC: 2000/mcL. All patients have stable arterial pressure. Antibacterial therapy upon starting the PMX: ceftazidime/avibactam, polymyxin, linezolid. Indications for starting PMX were as follows: endotoxemia due to Gram-negative bacteria, EAA more than 0.6 U (with WBC more than 1000/mkl), continuous fever, tachycardia, tachypnea, urination <0.5 mL/kg/hour, lactic acidemia, elevated PCT and CRP (to exclude immunological genesis of fever). CVC for hemodialysis were installed in all patients into femoral vein, and selective sorption was performed with Toraymixin B cartridge for, at least, 2 hours, in 2 procedures with 24-hour period.

Results

All children improved with reduced fever and stabilization of inflammatory markers, with diuresis of 2.5-3.0 mL/kg/hour. Standard posttransplant care was performed with antibiotics de-escalation. All patients are alive now. The follow-up period was 12 months in Pt. 1; 9 months in Pt. 2; 3 months in Pt.3.

Conclusion

Timely PMX-hemoperfsuion for selective sorption of endotoxin is associated with decreased risk of septic shock and lethal outcome after HSCT. We showed high importance of starting this therapy in order to avoid multiple organ failure. EAA in the patients with high WBC counts (>1000/mcL) is crucial to starting the hemoperfusion. Reduction of septicemia allows to continue standard posttransplant care.

Keywords

Oncology/hematology, pediatric, HSCT, sepsis, endotoxin, endotoxemia, hemoperfusion.

Intestinal syndromes are common after allogeneic HSCT, due to acute graft-versus-host disease (aGVHD) and/or infectious conditions. Later on, they may manifest as overlap syndrome combining the features of acute and chronic GVHD. Early reactivation of herpesviruses is a common finding after intensive cytostatic therapy and HSCT. The aim of our study was to assess detection frequency of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6) and herpes simplex virus (HSV) in colonic mucosal biopsies within first 180 days post-HSCT, as well as their possible association with common clinical complications.

Materials and methods

Our study group included 119 patients (1 to 72 years old) admitted to the R. Gorbacheva Research Institute for allo-HSCT from 2014 to 2020. Most patients were treated for acute myeloid leukemia (n=34), acute lymphoblastic leukemia (n=36), Hodgkin’s disease (n=10), severe aplastic anemias (n=15). The patients were subjected to haploidentical related (48%), allogeneic unrelated (41%), or allogeneic related HSCT (11%). Bone marrow or peripheral blood stem cells were transplanted in, resp., 42 and 58% of cases. Myeloablative conditioning was applied in 57% of cases. A total of 155 diagnostic forceps biopsies of colonic mucosa were taken during diagnostic fibrocolonoscopy in the patients with intestinal syndromes. The endoscopy was made in severe therapy-resistant intestinal syndromes. Intestinal GVHD or local infections were assessed by means of clinical criteria and biopsy histology. Most biopsies were taken from descending and sigmoid colon (52%) followed by transverse colon (25%), ascending colon (10%), caecum (5%), ileum (5%). All patients or their guardians have given their informed consent for medical procedures (HSCT and endoscopy). The samples of mucosa (3 to 6 specimens) were studied by standard histology with immunoassays for some viral antigens. DNA from colonic mucosa was monitored for EBV, CMV, HSV, and HHV type 6 A/B by means of commercial PCR test systems. The results were considered positive or negative, at a sensitivity of 400-1000 gene copies/mL (depending on the virus type). Statistical evaluation was performed by Statistica 10 software, using both parametric and non-parametric criteria.

Results

Positive HSV findings in colon mucosa were infrequent (up to 8% at 2-3 months post-transplant). High HHV6 incidence (a mean of 62%) was revealed over 6 months, whereas CMV reactivation was less common in mucosal biopsies (28-35%) within 4 months post-transplant. EBV incidence exhibited a significant increase, especially, at later terms (until 5-6 months post-HSCT). In available time-matched blood/biopsy pairs, the HHV6 detection rates in colon mucosa was sufficiently higher than in blood samples (resp., 59% and 18%, p<0.04). The CMV and EBV detection rates were similar in colon and blood samples. A significant correlation was found between blood and colon positivity for both CMV and EBV (r=0.489 and r=0.583; p<0.05). Looking for possible associations between positive viral findings in mucosal biopsies and patient characteristics, we did not reveal any significant correlations with most demographic and clinical features of the patients, including age, gender or primary diagnosis of the patients. However, we have revealed significant associations between EBV incidence and some post-transplant outcomes. E.g., higher EBV detection rates in colonic mucosa correlated with prolonged bone marrow engraftment in terms of delayed leukocyte and platelet recovery. Moreover, increased frequency of EBV-positive colonic biopsies was found in deceased patients with intestinal syndromes (resp., 56% versus 21%, p=0.02).

Conclusion

Significant correlation between EBV incidence in blood and gut biopsies suggests some relations between systemic and local EBV reactivation. Moreover, post-transplant hematopoietic reconstitution seems to be associated with local EBV reactivation, thus confirming a special role of EBV in post-transplant complications within 6 months post-transplant.

The study was supported by the Russian Science Foundation (Grant No. 22-15-00149 of 18.05.2022).

Keywords

Hematopoietic stem cell transplantation, colonic mucosa, biopsies, herpesviruses, detection rates, clinical associations.

Our aim was to describe the cases of rare invasive fungal disease (IFD), the features of the course and diagnosis of invasive fusariosis in high-risk patients.

Materials and methods

Laboratory diagnosis of IFD was carried out at the Department of Clinical Microbiology (Pavlov University) and included direct light and fluorescence microscopy of biological material with calcofluor white, as well as a cultural study of biological material obtained from patients. Inoculations were made on agar and Sabouraud broth and incubated at 28°C and 35°C until culture growth was obtained. Fungemia was diagnosed using Bactec blood culture flasks (Becton Dikinson). Pathogens were identified by morphological features, as well as by MALDI-TOF mass spectrometry (VitekMS, bioMerieux). The sensitivity to antimycotics was determined using the SenstitreYeastOne test system.

Results

From 2014 to 2022, invasive fusariosis was diagnosed in 3 patients who were treated at RM Gorbacheva Research Institute. Patient S, 17 years old, with hapten agranulocytosis and acute polysinusitis, necrosis of the nasal septum, lower and middle turbinates on the left, lateral wall on the left, and fistula of the paraorbital region on the left (August 2020). Microscopy of biopsy specimens revealed a narrow septate mycelium, branching at different angles, as well as structures resembling phialides with microconidia. Culture: Fusarium solani. Sensitivity – minimum inhibitory concentrations (MICs): anidulafungin >8 mcg/ml, amphotericin B 1 mcg/ml, flucytosine >64 mcg/ml, micafungin >8 mcg/ml, caspofungin >8 mcg/ml, posaconazole >8 mcg/ml, voriconazole 8 µg/ml, itraconazole >16 µg/ml, fluconazole >256 µg/ml. Conducted antifungal therapy – LC ampho B and surgical treatment. After cessation of hapten intake, agranulocytosis resolved and the patient was discharged in a satisfactory condition. Patient K., 46 years old, with progression of resistant AML and pneumonia (September 2020). Investigation of the BAL fluid SARS-Cov2 coronavirus (PCR) was detected, and microscopy revealed a narrow septate mycelium of the fungus with random branching and numerous swellings. Culture: Fusarium solani. MIC: anidul. >8 µg/ml, ampho. B >8 µg/ml, flucyte. >64 mcg/ml, mika. >8 µg/ml, caspo. >8 µg/mL, posa. >8 mcg/ml, vori. >8 mcg/ml, itra. >16 µg/ml, flu. >256 mcg/ml. Thus, the pathogen demonstrated resistance to all antifungals. Despite therapy, the patient died. Patient A., 20 years old, with progression of resistant AML, pneumonia and polysinusitis (August 2021). BAL microscopy revealed a wide non-septate mycelium, branching at a right angle – the mycelium of the Mucorales, however, no growth was obtained during culture. The patient underwent surgical treatment of pulmonary mucormycosis: right-sided pulmonectomy, resection of the ribs and soft tissues of the chest, as well as sinusotomy with the removal of necrotic tissues. Fusarium oxysporum was isolated during mycological examination of the biomaterial of the paranasal sinuses. MIC: anidul. >8 µg/ml, ampho. B at 1 µg/ml, flucyte. >64 mcg/ml, mika. >8 µg/ml, caspo. >8 µg/mL, posa. >8 mcg/ml, vori. 8 mcg/ml, itra. >16 µg/ml, flu. >256 mcg/ml. Antifungal therapy with LA ampho B was effective, but the patient died from the progression of the underlying disease. All patients had blood cultures, but no blood cultures were obtained from any patient.

Conclusions

Invasive fusariosis developed mainly in patients with a resistant course of the underlying disease. The paranasal sinuses were the main affected organ, no hematogenous dissemination was observed. Fusariosis caused by F.solani (FSSC) and F.oxysporum (FOSC) showed a high level of resistance to antifungal drugs. Microscopic signs of fusariosis in the biomaterial were non-specific and required mandatory cultural studies. Treatment of invasive fusariosis included antifungal therapy and surgical treatment; success depended on the control of the underlying disease.

Keywords

Invasive fusariosis, hematological and oncohematological diseases, paranasal sinuses, surgical treatment, resistance.

Coronavirus infection (COVID-19) caused by the severe acute respiratory syndrome virus, SARS-CoV-2, is among the most serious problems in the 21^st century, a threat to public health worldwide. The COVID-19 pandemic is still active in the vast majority of countries. Based on WHO statistics, children are less susceptible to COVID-19 than adults. However, pediatric patients with oncological and hematological diseases are at risk due to a pronounced weakening of immunity caused by chemotherapy or bone marrow transplantation. The objective of our study was to analyze the data of patients who received allogeneic hematopoietic stem cell transplantation (HSCT) over 2020-2022 and had a coronavirus infection, in order to assess the role of medical nurses in prevention of the infection spread, and in treatment of HSC recipients during the COVID-19 pandemic as well as to consider clinical cases of HSCT recipients who contracted the COVID-19.

Materials and methods

From January 2020 to September 2022, 400 allogeneic transplants of hematopoietic stem cells were performed at the Dmitry Rogachev Centre. Sixteen patients (14 diagnosed with ALL; 2, with acquired aplastic anemia) experienced coronavirus infection at early or late terms after HSCT. Two patients with acute lymphoblastic leukemia with positive PCR tests in bronchoalveolar lavage without clinical signs of COVID-19 still underwent allogeneic HSCT for urgent indications. During the pandemic, the center developed recommendations for the prevention of coronavirus infection, as well as introduced a special monitoring regimen for the COVID patients. The patients with positive PCR test were isolated in separate boxes with a special nursing station. The nursing shifts were arranged in compliance with individual staff protective measures. Thus, the nurses working with these patients did not contact with the COVID-free patients. In turn, the compliance with individual protection measures, separate staying of the nurses at the workplace, as well as timely vaccination, ensured the safety of medical personnel.

Results

During the COVID-19 pandemic, our center performed 400 HSCT, thus fully maintaining their activities. In the absence of clear recommendations for the management of immunocompromised patients with coronavirus infection, we were able to prevent the mass spread of infection, due to the rules developed inside the center and strict compliance with these measures. Only 4% of patients (16 cases before HSCT and 2, after HSCT) suffered with COVID-19.

Conclusion

The COVID-19 pandemic has become a challenge to medical staff, in particular, to nurses who daily provided care to the patients with acquired immunodeficiencies. Over 2.5 years, the nurses have studied recommendations for the prevention of COVID-19 spread, mastered new working skills, i.e., strict contact-aerosol isolation of patients, COVID-19 monitoring, work in the pre-hospital area and the sick patient’s boxes, monitoring the vital parameters in the patients. Nurses were also given an important role in explaining and clearly observing all cautions and hygienic rules to the patient caregivers.

Keywords

COVID-19, SARS-CoV-2, coronavirus infection, HSCT, HSC recipients, nurses.

Rare invasive mycoses are becoming an increasingly important medical issue in immunosuppressed patients. Emergence and improvement of new diagnostic methods led to identification of new pathogens. Among the Aspergillus genus, some species not related to Aspergillus (Asp.) fumigatus, Asp. niger, Asp. flavus and Asp. terreus were named “cryptic”. This group of cryptic fungi may represent clinical challenges due to their resistance to antifungals. The aim of our study was to describe patients with hematological malignancies with invasive aspergillosis (IA) caused by cryptic Aspergillae.

Patients and methods

From 2016 to 2021 years, 10 cases of probable cryptic IA were registered in CIC 725, according to EORTC/MSG 2020 criteria.

Results

Patients baseline characteristics included age, gender, primary disease, its clinical status, treatment modes (especially, HSCT). Dominant underlying disease was Non-Hodgkin lymphoma (n=4; 40%). The median age at the IA diagnosis was 34 years (10-66). Nine patients (90%) underwent hematopoietic stem cell transplantation (HSCT): 8 patients received allogeneic grafts (allo-HSCT), one patient had autologous HSCT. Allo-HSCT from HLA-matched related donor (MRD), match-unrelated donor (MUD), and haploidentical (Haplo) were performed in 3 (30%), 2 (20%) and 3 (30%) patients, respectively. The main causal pathogen was Asp. nidulans (n=4, 40%). Combination of Aspergillus species was observed in 2 patients (20%). Fungal co-infection was diagnosed in 3 patients (30%), i.e., 2 cases with Penicillium spp., and Lichtheimia ramose was revealed in one patient. Bacterial and viral co-infections were registered in 6 patients (60%). In all cases, the infection affected lungs (n=10, 100%). Clinical manifestations included fever in 5 patients (50%), cough (in 5 cases, 50%), and dyspnea reported in 3 cases (30%). The median term of IA development after HSCT was 309 days (35 to 2303). Primary antifungal prophylaxis included fluconazole (Flu) administered in 5 cases (56%), voriconazole (Vor) applied in 2 patients (22%), posaconazole (Pos), in 1 case (11%), and echinocandin (Echin), in 1 patient (11%). One patient received secondary prophylaxis with Vor. At the time of IA development, 3 patients (30%) received glucocorticosteroids for the treatment of severe GVHD. Frontline therapy included Vor (n=8, 80%), liposomal amphotericin B (Amph B) as monotherapy (n=1, 10%), or in combination with Pos (n=1, 10%). Second- and third-line therapies were required in 1 patient (10%), with IA due to Asp.calidoustus: Amph B + Echin and Amph B + isavuconazole. A single lethal case could be attributed to IA, this patient died on day 45 after IA diagnosis. Overall survival at 12 weeks was 90%.

Conclusion

Asp. nidulans is the most common agent causing IA by cryptic Aspergillus spp. No specific clinical symptoms and sites of lesions could be specified in these cases. Cryptic IA may often develop in combination with other fungal agents, together with bacterial and viral infections. In HSCT recipients, сryptic IA is a longitudinal post-transplant complication with, generally, good prognosis.

Keywords

Stem cell transplantation, Aspergillus, invasive aspergillosis, infectious complications.

In recent decades, there has been an increase in patients with invasive mycoses (MI) of the upper respiratory ways. This trend is due to increased numbers of patients with various risk factors, e.g., immunodeficiencies caused by prolonged use of glucocorticoids (GCS) and other immunosuppressive drugs in oncological and hematological diseases, allogeneic hematopoietic stem cell transplantation (allo-HSCT) followed by graft-versus-host disease (GVHD), HIV infection, prolonged massive antibacterial therapy, diabetes mellitus, bronchial asthma, prolonged staying at the intensive care units etc. Currently, higher MI occurrence may be assigned to pandemic of the new SARS-CoV-2 infection, secondary immunodeficiency conditions due to COVID-19 and its treatment. The purpose of the work was to study the features of risk groups, specific endoscopic and X-ray patterns in different lesions of nasal cavity and paranasal sinuses caused by various pathogens in the invasive mycoses, as well as to evaluate efficiency and features of surgical treatment in immunocompromised patients with sinus-orbital form of the disease.

Patients and method

Clinical analysis included 9 cases with IM observed at the R. M. Gorbacheva Research Institute (7 hematological patients over 2018 to 2021), as well as two patients who underwent COVID-19, treated at the Department of Otorhinolaryngology (Pavlov University) in 2021.

Results

The patients with MI had different background disorders, e.g., agranulocytosis after antitumor chemotherapy for acute leukemia (n=4), allo-HSCT (n=2) performed, resp., for acute leukemia and Hurler syndrome, and drug-induced hapten agranulocytosis (n=1), as well as usage of GCS for the COVID-19 treatment (n=2). The group included 3 males and 6 females at the age of 1.7 to 80 years, 3 children (33.3%) and 6 adults (66.7%). The main clinical manifestations of MI affecting upper respiratory tract were as follows: fever reaction >38°C (78%, only in hematological patients), nasal breathing disorder (100%), local hyperemia and facial edema (78%), pain/pressure and feeling of overflow in the facial area (78%), headache (44%), ptosis (22%), ophthalmoplegia (11%). Diagnosis of MI in 100% of patients included computed tomography (CT) of the paranasal sinuses, endoscopic examination, biopsy, direct microscopy, cultural examination of the biopsy. CT signs of MI included decreased pneumatization of sinuses (100%), destruction of bone tissue (78%). Sufficient lesions were revealed by endoscopy, i.e., necrosis of the mucous membrane (88.9%), destruction of bone structures (77.8%). The diagnosis of MI was established using microscopy with calcofluor staining of biopsy specimens (100%). Mycological studies in MI patients have revealed mucormycosis (n=4), fusariosis (n=2), aspergillosis (n=2), a combination of mucormycosis and aspergillosis (n=1). The patients received systemic antifungal therapy in accordance with international guidelines. Surgical treatment was performed in 100% of patients with mucormycosis and fusariosis as emergent care. Surgery in aspergillosis was carried out upon restoration of hematopoiesis.

Conclusion

Immunocompromised patients are at increased risk for development of invasive mycoses. Extremely rapid spread is a typical feature of MI, with development of significant lesions, e.g., tissue necrosis, destruction, bleeding, penetration into the orbit, or cranial cavity. The key efforts include early drug therapy and surgical intervention which is determined by a specific pathogen.

Keywords

Invasive mycosis, immunosuppression, oncohematological group, graft-versus-host disease.

The incidence of invasive fungal disease (IFD) with involvement of the maxillofacial region, nasal cavity and paranasal sinuses has increased over years. Traditional background immunodeficiency conditions were supplemented by post-COVID syndrome, a disorder complicating the SARS-CoV-2 infection – COVID-19, where glucocorticosteriod treatment (GCS) and high-flow oxygen therapy were used for a long time. Our aim was to evaluate the risk groups, course of the disease, diagnosis and surgical treatment of patients with IFD which affected the maxillofacial region.

Patients and methods

From 2010 to 2022, 6 hematological patients from RM Gorbacheva Research institute (5 children, 1 adult) and 12 patients treated at other clinics of the Pavlov University, were observed, including 11 post-COVID-19 patients with IFD of the maxillofacial region. The IFD diagnosis was based on objective examination of maxillofacial region, endovideoscopic examination of the nasal cavity, nasopharynx, bacteriological, mycological examination of affected tissues, cone beam computed tomography (CBCT) of dental and alveolar system, multi-slice computed tomography (MSCT) of the chest organs and facial portion of the skull, performed in angio-contrast mode, magnetic resonance imaging of brain, spirometry, examination by appropriate specialists. The pathogen identification was based on the microbial morphology, as well as on MALDI-TOF mass spectrometry with a VITEK MS instrument (bioMérieux), model 1.0.0.46. IFD was diagnosed according to EORTC/MSGERC criteria, 2020.

Results

The incidence of IFD affecting the bones of facial area and adjacent soft tissues in allo-HSCT recipients was less than 0.1% during the follow-up period. The aggravating conditions in hematological patients were as follows: agranulocytosis caused by antitumor chemotherapy for acute leukemia was observed in 2 patients (33%); allogeneic HSCT was performed in cases (50%) with acute leukemia and mucopolysaccharidosis type I (MPS I), and the drug-induced immune agranulocytosis has been developed in 1 patient after NSAID therapy. The following comorbidities were found in post-COVID syndrome: arterial hypertension, 92% (n=11); diabetes mellitus, 50% (n=6); chronic kidney disease 67% (n=4); acute liver failure (chemical poisoning), 8% (n=1). Clinical manifestations of IFD were as follows: disturbed nasal breathing, 100%; pain in the upper jaw, 89%; numbness of the jaw and face, 94%; ptosis, 39%; necrosis of the upper jaw mucosal membrane, 44%; swelling of the face and hyperemia, 50%; ophthalmoplegia, 28%; mycotic lung disease, 22%; odontogenic diseases, 50%. In one patient involvement of the lower jaw and neck was revealed; in 17, upper jaw was affected; in 5, zygomatic bone was involved; in 7, nasal structures; in 18, the paranasal sinuses. The CT signs of IFD were as follows: complete reduction of pneumatization of the single or both nasal sinuses, 100% of cases, destruction of the bones at the facial skull portion, 100%. IFD was confirmed by mycological examination in all the studied patients: mucormycosis (n=12), aspergillosis (n=3), a combination of mucormycosis and aspergillosis (n=3). Timing of diagnosis from the onset of local manifestations in the maxillofacial region was as follows: in hematological patients, 7±3 days; in the patients with post-COVID syndrome, from 40 to 270 days. In 83% of cases (n=15), systemic antifungal therapy was used, in accordance with international guidelines and with respect to drug sensitivity of the pathogens. Surgical treatment included necrectomy, resection of affected facial bones, 94% (n=17).

Conclusion

Invasive fungal diseases of the maxillofacial region, nasal cavity and paranasal sinuses are a rare complication in hematological patients, mainly, after allo-HSCT, more often in pediatric patients and occur at a frequency of <0.1%. However, a novel group of immunocompromised patients with post-COVID syndrome has emerged recently. Mandatory conditions for the successful treatment of mucormycosis are early diagnosis and antifungal therapy, surgical removal of affected tissues, control of the underlying disease, and decreased severity of immunosuppression. Surgical treatment leads to occurrence of defects that worsen the quality of life, e.g., requiring complex dento-maxillofacial prosthetics.

Keywords

Mucormycosis, maxillofacial necrosis, osteonecrosis, COVID-19-associated mucormycosis, САМ, COVID-M.

Mycoplasma is a common contaminant of cell cultures. Small size and resistance to many antibiotics prevents their timely detection and elimination. Mycoplasma, exhausts the nutritional medium, inhibits the in vitro cell growth, alters distinct properties of cultured cells (metabolism, proliferation, gene expression) thus making it difficult to use them in experiments as model lines and interpret the study results. In this regard, it is extremely important to control the presence of this pathogen in laboratories. The method of digital drop PCR allows this assays at high specificity and reproducibility level thus making it preferable in the absence of alternative assays. The purpose of this work was to adapt droplet digital PCR technique for detection mycoplasma contamination of cell cultures, as well as to screen for mycoplasma the available human cell lines at the laboratories of R. Gorbacheva Research Institute.

Materials and methods

Within the framework of this study, we tested the K562, Raji, Hek293, THP-1 cell cultures with suspected contamination. Their aliquots were thawed and cultured in appropriate media for 48 hours prior to sampling. The cells were then washed of the culture medium by centrifugation and resuspension in phosphate-buffered saline. Further on, whole genome DNA was isolated from the cells using spin column isolation technology (GeneJETGenomic DNA PurificationKit, ThermoFisher, USA). Concentration and quality of DNA preparation was measured using a Nanodrop device. To detect mycoplasma contamination, the MycoReal-Time (Evrogen) reagent kit was used, with detection of mycoplasma DNA by the RT-PCR method with TaqMan probes, but with the addition of the ddPCR^TM SupermixforProbes reagent kit specific to the digital droplet PCR (Bio-Rad, USA). The droplets were generated, and fluorescence was read using a QX200 AutoDGDropletDigital PCR System (Bio-Rad, USA) with automatic droplet generation.

Results

In order to adapt the MycoReal-Time (Evrogen) protocol to digital droplet PCR, the following combination of reagents per reaction was used: 10 µl ddPCR^TM Supermix for Probes, 5 µl 5X OligoMyco RT, 2 µl cell line DNA sample or MycoDNA Control. To avoid an overload of DNA-containing droplets, positive control samples were diluted 4 times relative to those recommended by manufacturer. The total reaction volume was 25 µl. PCR was carried out as follows: 95°C, 10 min; (95°C, 10 sec; 60°C, 1 min)x45 cycles. Fluorescence values were read in the FAM channel. Using this protocol, a positive control signal from the MycoReal-Time kit (Eurogen) was successfully detected. Screening of laboratory cell lines showed that three out of four analyzed cell lines (K562, HEK2963T and THP-1) were contaminated with mycoplasma. An attempt to eliminate K562 contamination by culturing them in the presence of clindamycin (900 μg/ml) and ciprofloxacin (120 μg/ml) for 14 days was successful, i.e., mycoplasma was not detectable by the digital drop PCR technique.

Conclusion

According to the results of digital droplet PCR, the MycoReal-Time kit protocol (Evrogen) was successfully adapted to the QX200 AutoDGDropletDigital PCR System (Bio-Rad, USA). This technique may be used on routine basis in the laboratories of R. M. Gorbacheva Research Institute for the control of mycoplasma contamination of cultured cell lines and primary cultures of human cells.

Keywords

Mycoplasma, contamination, cell cultures, DNA, polymerase chain reaction (PCR), digital drop PCR.

Some myeloablative total body irradiation (TBI)/total lymphoid irradiation (TLI) – based conditioning regimens prior to allogeneic hematopoietic stem cell transplantation are an effective treatment modality for some pathologic conditions. However, the issues of early and long-term toxicity, as well as some challenges in treatment planning and performance, are still a matter of debate. We aim to share our experience of interdisciplinary patient management in order to deliver these regimens. We also try to evaluate the method’s toxicity and effectiveness.

Materials and methods

From June 2018 to July 2022, 15 allo-HSCTs with TBI/TLI as part of the conditioning regimens were performed in a cohort of pediatric patients with refractory/recurrent B-ALL (n= 6, 40%) and T-ALL (n=7, 46.8%), JMML (n=1), AA (n=1). CNS involvement was registered in 40% of cases. The median age was 10 (5-15) years, mean age was 9.4 years. The median follow-up period was 24.2 (18 days-48 months) months. The TBI/TLI was carried out in LDC MIBS clinic using a linear electron accelerator “Varian Clinac 2100” in Rotational Radiation Techniques mode with volumetric modulation (VMAT). The total dose for TBI/TLI was 12 Gy (single fraction dose 2 Gy given twice a day) and 4 Gy (single fraction dose 2 Gy, once a day), respectively. A relative limitation for TBI was patient’s height of >160 cm, in which case the legs were bent in the knees and fixed with a vacuum mattress. Supportive therapy during the period of irradiation included infusion therapy and antiemetics.

Results

The following conditioning regimens were used: 11 cases (83.4%), TBI/Thiotepa/Fludarabine; 3 (16.6%), TBI/Melphalan/Fludarabine, and 1, TLI/Fludarabine/CyC/ATGAM. The donors were haploidentical in 12 (80%), MRD in 1 (6.7%), and MUD in 2 (13.3%) cases, respectively. GVHD prophylaxis included ATGAM/Rituximab/Abatacept in combination with TCR-aB depletion (60%), CyC/Tacrolimus/MMF (26.6%), Cyclosporine or Tacrolimus/MMF (13.4%). During the conditioning period we registered constitutional (14.3%) and neurotoxicity (14.3%), toxic hepatitis 1 grade (14.3%), and mucositis (64.3%). Infectious complications were registered in 100% of transplant recipients (80% caused by Gram-negative bacteria) with most common being enterocolitis (80%), CLABSI (13.3%), and UTI (6.6%). Reactivation of CMV infection was seen in 21.5% cases. The median time to granulocytes and platelets engraftment was 15 (9-20) and 19.5 (9-30) days, respectively. Acute GVHD was registered in 40% of cases with most common being cutaneous (28.6%) and intestinal (21.5%) forms, 1 case was refractory. The 2-years OS and EFS comprised 57.1% and 3-months HSCT-associated mortality was 21.4%. No long-term complications were registered during the indicated follow-up period.

Conclusions

TBI/TLI conditioning regimens were well tolerated with low incidence of early as well as delayed toxicity and are feasible in St. Petersburg hospitals. Based on results obtained it is possible to recommend the method in routine practice to patients with appropriate clinical indications. Further studies are needed in order to evaluate TBI-containing conditioning regimens effectiveness and toxicity in comparison to other regimens.

Keywords

Total body irradiation, allo-HSCT, children, hemoblastoses, radiation therapy.

Hematopoietic stem cell transplantation (HSCT) is one of the key stages in treatment of children with malignant and benign conditions. In some cases HSCT is the only radical treatment method possible. It is also used in rare malignant and non-malignant pediatric hematological diseases. In some cases these non-malignant diseases belong to the group of so-called precancerous states, which makes the topic relevant for specialists working with pediatric neoplasms. We present the N. N. Blokhin NMRCO experience of HSCT in children with rare oncological and hematological diseases.

Materials and methods

At the N. N. Blokhin NMRCO, a total of 17 pediatric patients (median age 47 months, range 25-192 months; M/F=8/9) with rare oncological and hematological diseases received HSCT for retinoblastoma (RB, n=3), germ cell tumor (GCT, n=8), Fanconi anemia (FA, n=2), dyskeratosis congenita (DC, n=3), and pleuropulmonary blastoma (PPB, n=1) in 2019-2022. The patients underwent autologous (auto-HSCT) and allogeneic (allo-HSCT) transplantations. All patients underwent pharmacological conditioning. In children with AF and DC (allo-HSCT) in included Busulfan, Fludarabine, ATG and Cyclophosphamide. In children with RB and GCT (auto-HSCT) it consisted of Etoposide, Thiotepa, Carboplatin and Cyclophosphamide, while in a patient with PPB (auto-HSCT) of Treosulfan and Melphalan. The HSC source was peripheral blood (PB) in auto-HSCT, and bone marrow (BM) from a sibling (n=4) or PSC from an unrelated donor (n=1) in allo-HSCT recipients.

Results

All patients successfully underwent HSCT. In early post-HSCT stages the following complications were registered: Gr 1-2 skin toxicity, Gr 1-2 oropharyngeal mucositis, Gr 1-2 neutropenic enterocolitis. These complications were successfully controlled by standard therapeutic interventions. Signs of Gr 1 acute GVHD were observed in 2 allo-HSCT recipients. Average time to engraftment after auto-HSCT was D+12, and D+19 after allo-HSCT. No significant toxicity was recorded. One death was observed in a child with RB 7 months after auto-HSCT due to relapse. The median follow-up is currently 9 (1-25) months.

Conclusions

HSCT is an acceptable option in children with rare oncological/hematological conditions. Each patient with such condition requires an individual approach to management during HSCT and subsequent follow-up.

Keywords

Hematopoietic stem cell transplantation, oncology, hematology, rare diseases, children.

While classical Hodgkin ‘s lymphoma is considered a curable disease and 80% of patients retain stable remission after first-line therapy, approximately 15% of them are primary refractory or develop relapse. Immune checkpoint inhibitors (ICIs) are effective in adults with Hodgkin’s lymphoma (HL), but there are only small pediatric groups described. Pembrolizumab and nivolumab are class IgG4 monoclonal antibodies to programmed death receptor 1 (PD-1). They selectively block interactions between PD-1 and its ligands PD-L1 and PD-L2, which restores the T-cells’ ability to recognize and destroy tumor cells. This study was aimed to evaluate the efficacy and safety of ICIs monotherapy in children and adolescents with relapsed or refractory HL.

Materials and methods

A total of 23 patients received monotherapy with nivolumab, and 2 patients with pembrolizumab. The median age was 15 (7-18) years. The HL morphologic variants were nodular sclerosis (n=20, 80%), HL with mixed cellularity (n=4, 16%), and HL with lymphocytes predominance (n=1, 4%). At the disease onset B-symptoms were registered in 14 (56%) patients, bulky disease (>7 cm) and extranodal lesions in 9 (36%) and 11 (44%) children, respectively. In 15 (60%) cases disease was primary refractory and in 10 (40%) the therapy was given at relapse. The median number of prior therapy lines was 3 (2-7). At treatment initiation 20 (80%) patients had disease progression, 4 (16%) partial response, and 1 (4%) stabilization according to the Lugano criteria. Nivolumab was given at dose of 3 mg/kg biweekly in 11 (48%) patients and as a single 40 mg dose every two weeks in 12 (52%) cases. Pembrolizumab was administered at a dose of 2 mg/kg every 3 weeks. Both drugs were given on outpatient basis. The median number of nivolumab injections was 8 (3-24). The response to the therapy was assessed according to LYRIC criteria. Upon ICIs course completion 11 patients received autologous and 1 allogeneic hematopoietic stem cell transplantation. In 2 (8%) cases, radiation therapy was used as consolidation.

Results

The overall response rate to one observed in adult patients and reached 88% (complete in 40% and partial in 48% of cases). With a median follow-up of 940 (107-2311) days all patients were alive, but PFS at 1, 2, and 3 years was 66%, 49%, and 24%, respectively. However, as patients responded to subsequent therapy, 15 (60%) are currently alive and in remission. Clinically significant complications (autoimmune thyroiditis and tuberculosis) were noted in 2(8%) patients. In case of thyroiditis it was effectively controlled by steroids and ICIs were then reinitiated. In case with tuberculosis the ICIs therapy was interrupted.

Conclusions

Checkpoint inhibitor therapy allows achieving response in the majority of children with primary refractory or relapsed Hodgkin lymphoma. However, as the response is then lost in most cases, some form of consolidation (e.g. autologous HSCT) may be recommended.

Keywords

Сhildren, Hodgkin’s lymphoma, refractory and relapsed disease, immune checkpoint inhibitors.

According to a 2003 WHO report, age is a socio-economic factor that impact on patients’ ability to follow doctor’s recommendations.

Aim

To demonstrate the effect of age on adherence among patients undergoing allo-HSCT.

Materials and methods

We analyzed 81 patients who entered the National Medical Research Centre of Hematology at the Russian Ministry of Health for allo-HSCT. The patients were diagnosed with acute myeloid leukemia (n= 48), and 33 patients were treated for acute lymphoblastic leukemia. The average age of the patients was 45 years, ranging from 18 to 63 years old. Adherence to treatment and medical care was studied using a specialized questionnaire KOP-25. The answers to each question were evaluated as point scores, then being summarized, and quantitative indices were calculated. On the basis of obtained scores, the indices of adherence were calculated having been expressed as conditional percentages. Differential indexes concerned adherence to the lifestyle changes, drug therapy, and medical care. We used scatter diagrams and regression analysis (LOESS method).

Results

The results of our study have shown similar reverse relationships between the patient’s age and adherence to the lifestyle (Cc) modification, drug therapy (Cd), and medical care (Cm). Moreover, we noted an age-dependent effect upon the integral index of adherence (С).

Conclusion

On the basis of the data obtained, we have concluded that the adherence for treatment shows a decrease for all tested indices with the patients’ age.

Keywords

Allogeneic HSCT, recipients, adherence to lifestyle modification, adherence to drug therapy, adherence to medical care.

Transfusions of blood components are an integral part of therapy for in pediatric patients with cancer and hematological conditions. Diagnosis, therapy type, previous treatment, performance status, hematopoietic stem cell transplantation type, and presence of AB0-incompatibility between donor and recipient etc. are among factors affecting transfusion therapy strategy. As overall number of blood transfusions is high, the medical personnel is tasked with providing each pediatric patient treated within bone marrow transplant clinic with an individual and safe approach to transfusions. This study aims to research the specific characteristics of pediatric blood transfusions in transplant clinical as well as at educating the nurses.

Materials and methods

Between 2018 and 2020 a total of 2104 pediatric patients with acute leukemia, malignant neoplasms, non-malignant hematological diseases, and inherited conditions were treated in RM Gorbacheva Research Institute. A total of 489 hematopoietic stem cell transplants (HSCTs) were performed with 327 of them being allogeneic and 162 being autologous. All patients received a total of 7898 of platelet concentrate (PC) transfusion during the study period, among them 2765 whole blood derived PC units, and 4647 apheresis PC units. PC transfusions (with maximum PC dose being 10 ml/kg/hour) were given if platelet count fell below 20*109 in presence of bleeding. Also, a total of 4417 red blood cell (RBC) units from matched donors were administered, 98.7% of patients received irradiated and leukoreduced red blood cells suspension. In 3754 (85%) cases transfusion were given as replacement in patients with mild to moderate (Hb counts of 70-80 g/l) clinically significant anemia. In this case the dose of 5, 7 or 10 ml/kg given art 2-5 ml/kg/hour was administered. In 663 (15%) cases transfusion were performed in patients with bleeding. In these cases the volume of RBC transfused depended on blood loss. All patients were monitored for blood pressure, heart rate, respiratory rate, body temperature, and diuresis before, during and after (within 2 hours) each transfusion with values obtained being recorded into patient’s sheet.

Results

No complications were observed during PC transfusions and 16 adverse events (ranging from urticaria to angioedema) were recorded after their completion and were treated by a physician with antihistamines and/or 1 mg/kg of intravenous methylprednisolone. No complications were observed during RBC transfusions with only one case of urticaria registered afterwards. All transfusions to patients weighing less than 30 kg were performed using Infusomat Space Line Transfusion BBraun systems. This allowed tuning to each patient’s individual characteristics appropriately changing transfusion volume and rate in order to avoid the volume overload, which may have disastrous consequences in children.

Conclusions

Individual approach blood components transfusion adopted to child’s characteristics and transfusion indications significantly reduces the post-transfusion complications risk. The properly trained team of nurses is required in order to provide safe transfusions and quality care to pediatric patients in bone marrow transplant clinic.

Keywords

Blood transfusion, red blood cell suspension, fresh frozen plasma, cryoprecipitate, donor platelets concentrate.

The main reasons for allogeneic hemopoietic stem cell transplantation (allo-HSCT) failure are relapses for malignant and graft failure/rejection for non-malignant conditions. However, the second allogeneic transplant may be a clinical option in these situations.

Materials and methods

The Sverdlovsk Regional Children Hospital Bone Marrow Transplant Unit was opened in 2006. By August 2022, a total 330 of allo-HSCTs were performed in 290 patients with different malignant and non-malignant conditions. Our study includes 18 pediatric patients aged 1 to 17 years who received second allo-HSCT due to underlying acute leukemia (n=12), CMML (n=1), acquired aplastic anemia (n=3), constitutional aplasia (n=1), and primary immune disorder (Nijmegen syndrome). In 7 cases, the second allo-HSCT was performed due to disease relapse, and in 11, because of graft failure/rejection. In half of the cases, the second transplant took place more than 6 months after the first HSCT.

Results

The underlying disorder in 7 relapsing patients with malignancies were ALL (4 cases); AML (1 patient), and CMML (1 case). The relapses were registered from 2 months to 2 years after the first transplant. All patients belonged to a high-risk group characterized by unfavorable disease biology and refractory clinical course. The presence of minimal residual disease (MRD) at the moment of allo-HSCT was the main risk factor for relapse. In all cases, the second transplant was performed from other donors and with another conditioning regimen (however, using myeloablative schedule). The following donors were recruited: haploidentical (n=5), HLA-matched unrelated donor (n=1), and HLA-matched related donor (n=1). The time from 1^st to 2^nd transplant exceeded 6 months in all cases (6.5 months to 2.5 years). One patient developed a refractory relapse and died due to disease progression, 6 patients are currently alive. Moreover, 6 patients with previously diagnosed leukemia received a second transplant due to graft failure/rejection. Three patients with complete donor chimerism received selected CD34+ boosts from the same donor without any conditioning regimen 40-60 days after the 1^st allo-HSCT leading to WBC engraftment in 2 cases. However, all 3 patients remained transfusion-dependent and died from complications (infections in 2, or TMA in 1 case). In 3 cases, a second allo-HSCT was performed from another donor after myeloablative conditioning. All patients were engrafted and achieved complete donor chimerism by the D+28. Two of these patients are currently alive (with 1.5 and 2 years of follow-up), one patient died with therapy-refractory relapse 5 months after the 2^nd transplant. Among 5 patients with non-malignant conditions, all second transplants were performed due to graft failure/rejection. The median interval between 1^st and 2^nd transplant was 2 months. In 4 cases, the same donors were used, with different conditioning regimen applied. In one patient with Nijmegen syndrome, a new donor was recruited. Four of 5 patients are currently alive and disease-free 6 months to 10 years after the second transplant. One patient with acquired aplastic anemia died of infection.

Conclusions

A gradual improvement in second allo-HSCT technique and supportive therapy makes it a feasible option for many patients. Its outcomes are mostly influenced by the disease status, patient’s performance score, and presence of infections/toxicity. The second transplant from haploidentical donor and subsequent immune- or chemotherapy may be an option for a number of high- or very high-risk patients.

Keywords

Children, leukemia, non-malignant conditions, hematopoietic stem cell transplantation, allogeneic, repeated.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains an important treatment option for patients with acute lymphoblastic leukemia (ALL). The results of allo-HSCT in children with ALL have significantly improved in the last decade. This study is aimed to evaluate the results of allo-HSCT in children, adolescents, and young adults with ALL at the Republican Scientific and Practical Center for Pediatric Oncology, Hematology, and Immunology.

Materials and methods

The study included 106 patients (75 children and 31 young adults; males, 59%) with ALL who received allo-HSCT at the Center for Pediatric Oncology, Hematology, and Immunology from 2001 to 2020. HLA-related, unrelated and haploidentical donors were used in 33, 59 and 8% of cases, respectively. Acute graft-versus-host disease (GVHD) was registered in 57 cases (54%); chronic GVHD, in 36 patients (34%). The Kaplan-Meier method was used to evaluate overall survival (OS), event-free survival (EFS), and GVHD and relapse-free survival (GRFS). In single-factor analysis survival was compared by Logrank test. The cumulative incidence of relapse (CIR) and death after allo-HSCT without recurrence (NRM) were calculated by the method of competing risks using Gray’s test. The results were evaluated and compared over two consecutive 10-year periods (2001-2010 and 2011-2020). The data was censored at 01.07.2022.

Results

The 3-year OS and EFS were, respectively, 42.5±7.8% and 40.0±7.8% for the first (n=40) and 76.7±5.3% (p<0.001), 68.1±5.8% (p<0.001) for the second (n=66) follow-up periods. The 3-year GRFS was 57.1±6.1% and 27.5±7.1%, respectively (p<0.001). The 3-year NRM rate over 2011-2020 was 9.2±3.6% (p<0.001), and CIR was 21.2±5.1% (p=0.60). There were no differences for aGVHD and cGVHD incidence between the both studied periods. The OS, EFS, and GRFS did not show statistically significant changes from 2001 to 2020 depending on donor type (related, unrelated or haploidentical), or age of the recipient (<4 years, 4-17, and >18 years). However, 3-year CIR in recipients <4 years (n=7) was 57.1±21.1% (p=0.01).

Conclusions

Thus, the outcomes of HSCT in children, adolescents, and young adults with ALL remain good when using traditional approaches. Development and implementation of cell therapy methods with their subsequent use in the treatment of children with ALL, and the active usage of transplantation from a haploidentical donor with appropriate support will further improve allo-HSCT parameters in children with ALL.

Keywords

Hematopoietic stem cell transplantation, acute lymphoblastic leukemia, children.

Nasogastric tube (NT) is a necessary tool for evaluation of the gastrointestinal tract and delivery of enteral nutrition (EN) in pediatric and adult patients. In the patients receiving treatment with chemotherapy and hematopoietic stem cell transplantation (HSCT), the indications for NT installation occur quite common, especially for implementation of EN and administration of medical drugs. At the same time, clinical characteristics of the HSCT patients, e.g., mucositis, severe thrombocytopenia, vomiting and diarrhea, require skilled nursing care and her competence in clinical nutrition and NT application. The aim of this work was to increase knowledge among the nursing staff concerning the nasogastric tube installation and providing enteral nutrition.

Materials and methods

We present our own experience of the NT usage and EN implementation, which was performed in accordance with general clinical nutrition recommendations for patients treated with chemotherapy and HSCT. From January 2020 to August 2022, 157 patients who required NT installation were included in the prospective study at the R. M. Gorbacheva Research Institute. This cohort included children 1 month to 17 years old (n=92, median age of 3.6 y.o.), and 65 adult patients (18 to 71 years old, median age of 33 y.o.). Most patients (n=108) underwent allogeneic HSCT (68.8%), 8.9% of patients were subjected to autologous HSCT (n=14), and chemotherapy was performed in 15.3% of the cases (n=20).

Results

NT was installed at the HSCT departments in 48.4% of the cases, as compared with 51.5% of the patients treated at the intensive care unit. The main indications for NT installation were as follows: EN, in 84.7%; drug delivery, in 3.8%; gastrointestinal paresis, in 6.3% of cases. In 32.4% of the patients, NT was installed upon initiation of mechanical lung ventilation. At the time of NT installation, such clinical manifestations as anorexia 83.4%, nausea 39.4%, vomiting 30.5%, diarrhea 33.7%, mucositis 20.3% (12.1%, of severe degree), intestinal and combined graft-versus-host disease were observed, respectively, in 5.7% and 8.2% of cases. In 42.6% of cases, NT was installed in presence of thrombocytopenia IV (<20×10⁹/l), which, however, did not lead to nasal bleedings. The duration of the NT usage ranged from 1 to 118 days (a median of 11 days). The most common reasons for NT removal were, e.g., restoration of self-feeding in 50 patients (31.8%); no further need for NT (n=59, 37.6%) and self-removal of NT by the patient (n=22, 14% of cases). Acute complications during NT installation were registered in 3.2% of cases manifesting as nasal bleeding (n=4) and NT dislocation (n=1).

Conclusions

NT installation and assisted EN are fairly frequent clinical procedures in the patients undergoing chemotherapy and HSCT, which can lead to complications in cases of wrong implementation of this technique, thus requiring theoretical knowledge and practical skills from the nurses.

Keywords

Nasogastric tubes, enteral nutrition, nursing care, cancer therapy.

Virtual technologies are effective in rehabilitation of patients with different conditions including hematology and cancer department patients. The early physical rehabilitation may be effective in allo-HSCT recipients allowing shorter post-transplant recovery period.

Patients and methods

We evaluated early physical rehabilitation effect in pediatric bone marrow transplant recipient. We have also assessed effectiveness of virtual technology-based rehabilitation in a child with post-chemotherapy polyneuropathy.

Results

Case 1: Patient D., 12 years old, was diagnosed with acute lymphoblastic leukemia, post-transplant period after the second allogeneic HSCT. Movement disorders were the most prominent symptoms at the beginning of treatment. Rehabilitation proceeded for 20 days including bicycle kinesis, coordination gymnastics, manual stretching. The therapy resulted into an increase in limb muscle strength from an average of 3 to 4 points (MRC test), an increase in mobility (Rivermead test from 7 to 8 points), a reduction in the risk of falling (Berg test from 42 to 49 points), complete movement range recovery. Case 2: Patient K., 8 years old was diagnosed with chemotherapy-induced peripheral polyneuropathy of lower extremities manifesting with lower moderate-degree paraparesis being most evident on the right side, balance disturbance and motor walking stereotype due to kinesiophobia. Rehabilitation course lasted for 2 weeks, 1 procedure in 3 days. It resulted into walking without an auxiliary device, an increase in muscle strength from 3 to 4 points on the MRC scale, the Berg balance scale from 35 to 42 points from 35 to 42, an increase in walking speed (from 0.07 to 0.19 m/s), a decrease in anxiety.

Conclusion

The proposed therapy complexes in these patients were adequate and made it possible to achieve the goals of rehabilitation.

Keywords

Early physical rehabilitation, virtual technologies.

Haematopoietic stem cell transplantation (HSCT) has now proven effective in the treatment of blood diseases, a number of immune diseases and some types of solid tumors. However, it is worth of note that the nursing care in the post-transplant period is an important factor influencing the further quality of life of the recipient after HSCT.

Patients and methods

At the Republican Scientific and Practical Centre for Child Oncology, Haematology and Immunology, 32 allografts and 18 autografts were performed in 2021. Over 7 months of 2022, 10 allografts and 12 autografts were performed. The most frequent complications were toxic dermatitis, mucositis, and enterocolitis. The duties of a nurse of the transplantation department include constant monitoring of vital symptoms observed in the patient (heart rate, blood pressure, respiratory rate, oxygen saturation), providing assistance in hygienic procedures, performing local treatment of skin and mucous surfaces, one may perform laser therapy of oral cavity in cases of oral mucositis, as prescribed by the attending physician, control of administered medications, providing care for central venous catheters and nasogastric tubes, urinary catheters. All manipulations are carried out under the conditions of strict sterility and aseptics which should be permanently observed.

Conclusion

Due to well arranged and high-quality work of the transplant nurse the risk of post-transplant complications could be sufficiently reduced.

Keywords

Hematopoietic stem cell transplantation, complications, nursery.

Due to the restricted capacity of resident chondrocytes to regenerate injury of cartilage, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. The aim of this work was to develop the method for injection of MSCs in the treatment of osteoarthritis.

Materials and methods

MSCs have been derived from bone marrow and expanded in vitro in DMEM with 10% FBS-MSC. Chondrogenic pre-differentiation of MSCs in 2D were obtained by culturing in DMEM with 10 ng/ml TGFβ3, 100 ng/ml IGF, 100 nM dexamethasone (Life Technologies, USA), 1% antibiotic for 7 days. The injectable implant was obtained by mixing predifferentiated MSCs with 1% hyaluronic acid in a ratio of 1:1. Chondrogenesis of MSCs was confirmed by immunofluorescence staining, PCR. The clinical efficacy of injectable implants was assessed by VAS (Visual Analogue Scale) and WOMAC (Western Ontario McMaster Universities OA Index).

Results

The study included twelve patients with osteoarthritis at the 2-3^rd severity stage. The cells had high 98 (97.5; 99.5)% viability, phenotype of MSCs (more than 95% of cells had CD90, CD105, and CD73). Chondrogenesis after supplementation with the growth factors was proven by detection of the type II collagen and aggrecan synthesis by MSCs, as shown by immunofluorescence. A change in morphology MSCs from spindle-shaped to “cobblestone-like” was detached. We have shown an increased expression of COL2 by 10 times (p=0.002), COL11 by 3 times (p=0.03), along with 5-fold decrease of COL1 expression (p=0.03). A total of 12 clinical preparations of chondrogenic pre-differentiated MSCs with 1% hyaluronic acid were obtained, at the average number of 31.0 (25.0; 37.5)×10⁶ cells in a mean volume of 0.95 (0.65; 1.05) mL. The cellular implants were injected to the patients intra-articularly. After a follow-up periods of 6 and 12 months, clinical effects were evaluated by means of VAS and WOMAC assessment scales. By the VAS scale, distinct changes were shown at the end of follow-up periods, i.e., from 5 cm (4; 6) to 2.5 cm (2; 3.5), and to 2 cm (1; 4), respectively (p <0.05). The data of WOMAC evaluation score showed improvement during the follow-up periods from 56.5 (37; 63) to 31 (22; 41) points, and to 31 (9; 48) respectively (p <0.05). Systemic or local complications were not observed in the patients after injections of implanted cells into the knee joint.

Conclusion

The results of this study confirm the efficacy of intra-articular injections of chondrogenically predifferentiated allogeneic MSCs in hyaluronic acid when treating patients with osteoarthritis.

Keywords

Mesenchymal stem cells, cell-based therapy, osteoarthritis.

Chimeric antigen receptor (CAR) T-cell therapy has been actively introduced into clinical practice in Belarusian specialized healthcare institutions since 2020 as a part of research protocol NCT05333302. The main objective of given research protocol is safety and efficiency evaluation of locally manufactured CAR T-cell product in the treatment of CD19-positive relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) and lymphoma.

Materials and methods

Manufacture and clinical application of CAR T-cell product was performed at N. N. Alexandrov National Cancer Center of Belarus and in Belarusian Research Center for Pediatric Oncology, Hematology and Immunology. Populations of CD4 and CD8 T-cells were obtained by separate procedures from the apheresis product by immunomagnetic selection. Genetic modification of T-cells was performed by lentiviral transduction with bicistronic vector FMC63-41BB-3z-P2A-hEGFRt coding for second generation CAR and truncated variant of human epidermal growth factor receptor (hEGFRt). After ex vivo cell expansion for 12-14 days, the final CAR T-cell product underwent quality control including sterility check, immunophenotyping and functional activity testing. Pre-conditioning regimen of lymphodepleting chemotherapy included fludarabine and cyclophosphamide. Dose of infused CAR T-cells was in the range of 1-3×10⁶ cells per kilogram of body weight depending on tumor burden. Treatment of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (ICANS) included tocilizumab and corticosteroids.

Results

CAR T-cell therapy was carried out for 13 patients. CAR T-cells expansion in vivo observed in 12 patients with median peak value 17 (range 1.7-224) cells/mcL of peripheral blood (PB). Median persistence of CAR T-cells in PB of recipients was 60 days (range 13-90). B-cell aplasia was observed in all patients with proven expansion of CAR T-cells. CRS events were confirmed in 77% of cases: grade 1, 7 patients; grade 2 CRS was seen in 1 case; grade 3, in 2 patients. ICANS developed in 46% of cases: grade 1, in 5 patients; grade 3, in 1 case. Complete response (CR) was detected in 6 out of 8 cases with B-ALL. One patient did not respond and died due to disease progression. One more patient died due to complications (CRS + sepsis) not achieving clinical response. Three out of six patients with CR subsequently developed relapse after 4, 5 and 18 months correspondingly. Median remission duration in given group was 9 months (range 4-18). CR was detected in 3 out of 5 patients with B-cell lymphomas, partial response (PR) in 1 case and 1 patient died due to complications (CRS + sepsis) not achieving clinical response. Patients with CR are in remission for 1, 6 and 10 months correspondingly. Patient with PR is preparing for autologous hematopoietic stem cell transplantation. Outcome: of 13 treated patients 9 are alive, 4 patients are dead, median duration of observation was 6 months (range 1-20).

Conclusion

The CAR-T cell technique is safe, reproducible and effective upon current follow-up of ALL patients.

Keywords

CAR Т-cell therapy, B-ALL, B-cell lymphoma.

Primary resistance to CAR T-cell therapy occurs in 10-20% of pediatric B-ALL cases [PA Atilla, 2022]. Impaired functional activity of CAR T-cells and biological properties of leukemic cells are among the causes of described phenomenon. Anti-CD19 CAR T-cell therapy was carried out at Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (BRCPOHI) for 8 B-ALL patients with one case proven to have primary resistance to the given therapy.

Materials and methods

A 7-year old boy was diagnosed with B-ALL, positive for MLL (KMT2A) gene rearrangement (MLL-AF9), BIII immunophenotype positive for NG2, CD33, CD65 and CD15 expression. Short-term remission was achieved after treatment according to ALL-MB-2015 protocol with subsequent isolated bone marrow (BM) early relapse I. Leukemic cells were still positive for MLL-AF9, but immunophenotype changed to BI with CD33 and CD15 expression. Anti-relapse treatment failed to induce remission. Moreover, leukemic clone acquired multi-drug resistance to chemotherapy. The decision was made to perform anti-CD19 CAR T-cell therapy. Taking into account high tumor burden (93% of blast cells in BM) and patient general condition, final dose of CAR-T-cells (1×10⁶ cells/kg) was given as fractionated infusions of 10% and 90% over 7 days. Immune monitoring did not reveal significant CAR T-cell expansion in recipient BM or peripheral blood (PB). Peak concentration of CAR T-cells in patient PB was 0.05 cells/μl, while median value in group of 7 patients with noticed expansion was 17 cells/μl [range 1.7-224]. Despite carried out immunotherapy, growth of leukemic blasts’ count was observed in PB and BM. Immunophenotyping of leukemic cells at this stage revealed mixed phenotype (B/My). Patient died of the disease progression 30 days after the last injection of CAR-T-cells.

Results

It is well known that efficiency of CAR T-cell therapy depends dramatically on cell product characteristics. High content of exhausted and terminally differentiated T-cells significantly reduce their potential for expansion and persistence in vivo. In described case number of CCR7-positive CD4 and CD8 T-cells in final product was 84% and 90% correspondingly. Moreover, CAR-T-cells demonstrated high in vitro cytotoxicity against model cell line and recipient leukemic blasts. It was previously reported that multi-lineage phenotype (CD19, CD20, CD33, CD34) is frequent in CAR T-cell therapy non-responders [KE Masih, 2022]. It must be noticed, that B/My phenotype was also observed in described patient. Another one mechanism of primary resistance to CAR-T-cell therapy is CD58 gene mutations or loss of corresponding protein expression on tumor cells [RG Majzner, 2020, Xin Yan, 2022]. CD58 protein is a ligand for T-cell costimulatory receptor CD2. Disruption of CD58 interaction with its partner CD2 leads to formation of suboptimal immunological synapse, which results in impairment of CAR T-cell functional activity and loss of its expansion potential. Normalized to healthy lymphocytes, coefficient of CD58 expression on the surface of given patient blasts was 0.95. Whereas median value for the same parameter in group of CAR T-therapy responders was 2.46 (range 1.2-6.56).

Conclusions

We suppose that the described case of primary resistance of B-ALL to anti-CD19 CAR T-cell therapy can be explained by patient’s leukemic cells properties: mixed immunophenotype and rearrangement of MLL gene, decreased expression/loss of surface CD58 antigen.

Keywords

Total body irradiation, allo-HSCT, children, hemoblastoses, radiation therapy.

The active use of genetically modified natural killer (NK)-cells is limited by methodological difficulties of delivering genetic material to these cells. Lentiviral transduction is one of the cell modification methods. However, its use is limited by low yield of transduced cells. The virus derived from the VSV-G vector cassette, typically used to produce CAR-T cells, doesn’t efficiently transduce NK-cells. To increase the efficiency of NK-cell transduction, we studied alternative pseudotyped proteins. The pseudotyped lentiviral baboon envelope vector BaEV-TR shows higher transduction efficiency. It binds the entry receptors of the sodium-dependent neutral amino acid transporter ASCT-1 and ASCT-2 and is widely expressed in hematopoietic lineages. Thus, the use of alternative pseudotypes of lentiviruses will provide solution of many fundamental issues and specify the tools of obtaining sufficient numbers of genetically modified NK cells. Our objective was to compare the efficiency of NK-cell transduction using two variants of lentiviral particles based on the BaEV-TR and VSV-G vector cassettes.

Materials and methods

We used peripheral blood mononuclear cells from two healthy donors. A primary culture of NK-cells was obtained using a CD3-depletion kit, after which the cells were cultured in PRMI-1640 complete growth medium supplemented with IL-2 500 IU, 72 hours before transduction; two lentiviral vectors with different envelope plasmids were also used; in the first variant, pMD2.G was used, encoding the surface glycoprotein G of the vesicular stomatitis virus (VSV-G); in the second, BaEV-TR, encoding the modified envelope glycoprotein of the baboon retrovirus. The psPAX2 plasmids, containing gag-pol and pUltra genes which encode the EGFP reporter protein gene, were included in both variants. NK cells were grown in complete RPMI medium prior to transduction. The multiplicity of infection was of 1, 5, 10. After transduction of the virus, the cells were incubated at 37°C, and the medium was changed after 24 hours. The level of transduction was determined by flow cytometry by the expression of the EGFP reporter protein after 72 hours.

Results and discussion

We compared the transduction efficiency of primary NK cells with two variants of lentiviral particles based on the BaEV-TR and VSV-G vector cassettes. The percentages of transduced cells at the infection multiplicity of 1, 5, 10 with VSV-G and BaEV-TR were 0.28%, 1.5%, 2.8% and 50%, 62%, 70%, respectively. The VSV-G receptor is a low-density lipid receptor LDL-R which is not expressed in either resting or activated NK-cells [1] thus explaining the low transduction rate we obtained. However, the baboon envelope receptors (ASCT-1 and ASCT-2) are overexpressed in response to activation of NK-cells by IL-2, IL-12 and IL-21, thus leading to increased efficiency of BaEV-TR transduction [Bari et al., 2019] as confirmed by our results.

Conclusions

The percentage of NK cells transduced with a lentiviral vector employing BaEV-TR vector cassette is higher than with a VSV-G-based vector cassette, which may be further used for immunotherapy with modified NK cells.

Reference

Bari R [et al.] (2019) A distinct subset of highly proliferative and lentiviral vector (LV)-transducible NK-cells define a readily engineered subset for adoptive cellular therapy. Front. Immunol. 10:2001. doi:10.3389/fimmu.2019.02001

Keywords

NK cells, transduction, lentivirus, pseudotyped envelope particles, retrovirus, baboon.

Arthritis is a heterogeneous group of diseases that affect one or more joints and are caused by inflammatory damage of various genesis, by involving the synovial membrane, capsule, cartilage and other components of the joint. Inflammatory diseases of the joints (IDJ) occur in 0.3-3% of the population worldwide. In particular, they affect 21% of adults in Russia. The joint swelling, stiffness of movements and chronic pain are observed in arthritis, thus altering normal working capacity, and often leading to disability. Development of novel drugs for the treatment of arthritis is an urgent task, due to variety of the IJD lesions and common inefficiency of basic, biological and glucocorticosteroid therapy. Radiopharmaceuticals show a number of advantages over radiation and chemotherapy used in the IDJ treatment. Hence, the aim of the study was to develop a radiopharmaceutical of a complex composition and evaluate its therapeutic effect in the animal model of arthritis.

Materials and methods

The ¹⁸⁸Re-PLA-MPs preparation was developed in the form of ¹⁸⁸Re-labeled polymer carriers. Basic properties of the carriers, their toxicity and safety were assessed, as well as the drug biodistribution. The results of the drug stability testing and its retention at the injection site were registered by SPECT and PET-CT images taken on the 1^st, 2^nd, 3^rd and 7^th day of the study, as well as by direct radiometry with measurement of the radioactive signal (%ID/g). Therapeutic efficacy of the radiopharmaceutical was evaluated in a model of inflammatory disease of large joints in the Wistar rats by measuring changes in the joint diameter and histology examination. The data on expression of proinflammatory cytokines were found to be consistent with histological data.

Results

The biodistribution experiments have shown that 98% of the radioactive drug remained at the injection site, without penetrating healthy tissues. Biological effects of the drug in the IDJ treatment was shown by means of the joint sizing, measurements of inflammatory blood cytokines and histological analysis.

Conclusion

According to experimental results, the developed radiopharmaceutical has shown its efficiency in the animal model of inflammatory joint disease produced in Wistar rats, thus providing a basis for further studies in this area.

Keywords

Arthritis, synovectomy, microcarriers, radionuclide therapy, rhenium.

Gene therapy based on transplantation of genetically modified hematopoietic stem and progenitor cells (HSPCs) using engineered nucleases (ZFN, TALEN, CRISPR/Cas9) has a strong potential for the treatment of a number of human monogenic diseases. The basis of this treatment method is the insertion of a healthy copy of a DNA segment or an entire transgene into the locus of a double-strand break formed by a nuclease, delivered as a donor template in various forms. It is known that an important factor affecting the insertion efficiency is the structure and length of the donor DNA template introduced into the cell. Among various forms (single- or double-stranded DNA, circular or linear, viral or non-viral, etc.), linear double-stranded linear DNA is of interest for preclinical development of gene therapy cell products due to potential efficiency and robust production. The limiting factor in their use is toxicity to primary human cells, which depends on the structural parameters of the templates [Paludan et al., 2013; Kath et al., 2022]. The aim of this work is to optimize the structure of the donor template introduced into the cell in the form of a linear double-stranded DNA used for homologous repair of TALEN-mediated targeted DNA breaks in order to increase the efficiency of editing/inserting therapeutic transgenes in human cell lines, which are the closest models of HSPC.

Materials and methods

Synthesis of variants of donor linear DNA templates was performed by PCR from plasmid DNA encoding the GFP transgene and homology domains to the CCR5 locus at the site of the formation of a 400 bp long double-stranded break. Transfection of K562 cells was carried out at a concentration of 3.3*10⁶ cells/mL according to a previously developed two-stage electroporation protocol using a Gene Pulser Xcell System electroporator (Bio-Rad, USA). The mRNA concentration during transfection was 25 µg/mL. The concentration of donor templates was 20 μg/mL. The following modifications of donor templates were studied: the addition of a sequence that inhibits sensors of intracellular immune response to exogenous DNA or damage to genomic DNA (TLR9/AIM2/cGAS) – A151 to the sequence of the donor template, as well as stabilization of the ends of the donor template with phosphorothioate bonds. The efficiency of transgene insertion and cell viability were assessed by flow cytometry.

Results

A two-stage transfection protocol was developed, which provides up to 85% knockout efficiency of the target CCR5 gene with cell survival from 30.4 to 46.2%. The transfection efficiency of the donor template in the form of a double-stranded linear DNA molecule was 7.1-23.6%. According to flow cytometry data, the efficiency of transgene insertion by the sixth day after transfection was 6.9-13.2%, depending on the type of modification of the ends of the donor template, and was maximal for the donor template without modifications. In comparison with the previously obtained preliminary data and control samples in the current experiment using plasmid DNA as a donor template, the efficiency of transgene insertion was higher when using linear DNA templates – 2.6% vs. 13.2%.

Conclusion

The development of linear double-stranded DNA templates as donors for the repair of precised DNA breaks holds promise for the development of gene therapy cell products for the treatment of monogenic human diseases.

The authors are grateful for financial support from the Russian Foundation for Basic Research, grant No. 19-29-04025mk.

Keywords

Gene therapy, nonviral, gene editing, TALEN platform, CCR5 gene.

Current studies provide data on the involvement of NCSTN gene encoding nicastrin (the major subunit of γ-secretase), in various neoplastic processes. It has also been shown to play a role in regulation of immune cells. Therefore, the study on the functioning of this gene may provide an opportunity to understand the mechanism of neoplasia in different tissues, and to reduce the risk of malignancies in patients with bone marrow dysfunction.

Materials and methods

At the beginning of this study, each of the gene isoform annotations was compared with mRNAs and ESTs data to select the annotated version. After this analysis, we may state that each annotation has isoforms carrying extra information about the gene, e.g., additional exons which are not mapped in mRNAs and ESTs, or absence of protein-encoding exons. These data were further analyzed for ability of this gene to encode proteins, transcription, expression, and interaction with proteins.

Results

The NCSTN gene is present on the chromosome 21. It includes 17 exons and 16 introns being located on the plus chain, thus indirectly indicating to its protein-encoding function. The gene is unique due to the presence of repeats only in introns, there are no pseudogenes, according to the BLAST database. The gene is annotated correctly, and its structure is true, since the peaks of the transcription track coincide with exons of the annotated isoforms. When examining the gene for protein-coding ability, we may conclude that, for each exon, the signal translational peaks obtained experimentally correspond to the annotated protein-coding regions of the gene. Most peaks of the ribosomal profiling correlate with translated peptides, thus presuming this gene to have a protein-coding function. Analysis of the gene by RNA-seq track data and CAGE peaks was based on the results obtained in A549 cell culture. When analyzing the RNA-seq data, we may suggest that 65.4% of RNA remains in the nucleus and 34.6% is released to the cytoplasm. According to the CAGE data, 72.4% of RNA remains in the nucleus and 27.6% migrates to the cytoplasm. The presented RNA-seq and CAGE data were compatible, i.e., the cytoplasm contains protein-translating RNA. Examination of the gene for mutations in the OMIM Genes database indicates that the gene has a mutation and can cause autosomal dominant inherited disorders manifesting by familial acne, familial purulent hydradenitis with bone marrow dysfunction. The encoded proteins cleave integral membrane proteins, including Notch receptors and beta-amyloid precursor protein, thus, probably, being a stabilizing cofactor required for assembly of the gamma-secretase complex. Similarly, according to the Gene Interactions track, this gene controls some protein-protein interactions that would be disrupted by gene mutations in absence of a final functional protein product.

Conclusion

Upon the data analysis, we may conclude that the point mutations of the gene, including those found in tumor samples, coincide with the coding sequences of exons. Moreover, the COSMIC Regions database contains information on the point mutations of this gene found in tumors, e.g., malignant melanomas, adenomas, carcinomas, sarcomas, gliomas, and in lymphoid neoplasms. These disorders arise not only due to point mutations in the coding gene sequences, as displayed in the COSMIC Regions, ClinVar Short Variants, and HGMD Variants databases, but also occur due to deletions and duplications affecting the entire length of this gene, as displayed in the ClinVar Long Variants and Development Delay databases.

Keywords

Bone marrow, transcriptomics, NCSTN gene.

Collaboration between bone marrow donor registries of different countries increases the chances of HLA-compatible unrelated donor selection for patients in need of allogeneic hematopoietic stem cell transplantation without available donors in registries of the country of residence. The donor search success and duration depends on patients’ HLA-haplotypes frequency, as well as patient’s ethnic group representatives in the registry. We performed a comparative analysis of the profile of HLA-A*-B*-C*-DRB1*-DQB1*-haplotypes of potential hematopoietic stem cells (HSC) donors of the FSBI RosNIIHT FMBA of Russia and the Republic of Belarus registries.

Materials and methods

The study included 2853 potential HSC donors of FSBI RosNIIHT FMBA of Russia registry. Primary immunogenetic examination was performed using PCR-SSP (“Protrans”, Germany) and PCR-SSOP (“BAG HEALTH CARE”, Germany). The surveyed group of potential HSC donors of the registry of the Republic of Belarus included 2420 people. PCR-SSOP (“Immucor”, USA) and PCR-SSP (“One Lambda”, “Olerup”, USA) were used for primary HLA-typing of donors. The HLA haplotypes frequencies were determined by maximum likelihood method using the EM algorithm provided by Arlequin 3.5 software.

Results

A total of 1702 HLA-haplotypes were identified in the FSBI RosNIIHT FMBA of Russia cohort examined. The most common HLA-haplotypes were: A*01-B*08-C*07-DRB1*03-DQB1*02 (0,0366), A*03-B*07-C*07-DRB1*15-DQB1*06 (0,0269), A*03-B*35-C*04-DRB1*01-DQB1*05 (0,0238), A*02-B*13-C*06-DRB1*07-DQB1*02 (0,0204), A*02-B*07-C*07-DRB1*15-DQB1*06 (0,0184), A*25-B*18-C*12-DRB1*15-DQB1*06 (0,0127), A*02-B*18-C*07-DRB1*11-DQB1*03 (0,0126), A*02-B*15-C*03-DRB1*04-DQB1*03 (0,0123), A*02-B*41-C*17-DRB1*13-DQB1*03 (0,0109), A*30-B*13-C*06-DRB1*07-DQB1*02 (0,0099). Also, 1365 HLA-haplotypes were determined in the Republic of Belarus potential HSC donors registry. The most common HLA-haplotypes were: A*01-B*08-C*07-DRB1*03-DQB1*02 (0,0445), A*03-B*07-C*07-DRB1*15-DQB1*06 (0,0330), A*02-B*13-C*06-DRB1*07-DQB1*02 (0,0294), A*02-B*07-C*07-DRB1*15-DQB1*06 (0,0194), A*03-B*35-C*04-DRB1*01-DQB1*05 (0,0179), A*02-B*18-C*07-DRB1*11-DQB1*03 (0,0159), A*25-B*18-C*12-DRB1*15-DQB1*06 (0,0141), A*02-B*57-C*06-DRB1*07-DQB1*03 (0,0118), A*11-B*35-C*04-DRB1*01-DQB1*05 (0,0112), A*23-B*44-C*04-DRB1*07-DQB1*02 (0,0109), A*02-B*27-C*02-DRB1*16-DQB1*05 (0,0105). The comparative analysis revealed similarities as well as differences in HLA-haplotypes distribution among donors of Belarusian and Russian registries. In particular, the frequencies of A*02-B*13-C*06-DRB1*07-DQB1*02, A*02-B*57-C*06-DRB1*07-DQB1*03, A*02-B*27-C*02-DRB1*16-DQB1*05 haplotypes are significantly higher in donors of Belarus Republic registry (p=0.04; 0.01 and 0.01 respectively). The frequency of A*02-B*41-C*17-DRB1*13-DQB1*03 haplotype is considerably higher in donors of FSBI RosNIIHT FMBA of Russia registry (p=0.03).

Conclusions

The HLA-A*-B*-C*-DRB1*-DQB1*-haplotypes profiles of potential Belarusian and Russian registries HSC donors have characteristic features. Using united donor resources for donor search will increase patient’s chances for optimal unrelated donors selection in term of their immunogenetic characteristics.

Keywords

Donor registry, hematopoietic stem cells, HLA-haplotypes.

As multinational Russian population consists of people with vastly different HLA phenotypes, the bone marrow donors’ registry should include as many donors from local populations as possible. Each year Republic of Sakha, Yakutia (RS, Y) 5-6 adults and 2-3 pediatric candidates for hematopoietic stem cell transplantation (HSCT) are registered by hematologists, and the number of patients with hematopoietic and solid tumors potentially requiring transplant gradually increase. The HLA alleles frequencies in Yakutia differ significantly from those observed in most donors from Russian population (Fefelova V.V., 2015]. However, this data may help to form a Registry for potential unrelated donors based on HLA combinations most often seen in HSCT recipients from Yakutia making the matched donor occurrence more probable. While general probability of donor selection in overall Russian Federation registry is 1 in 50000, the national Registry provides 1 potential donor in 5000.

Materials and methods

According to 2010 census the Russian population consists of more than 80% of Russians, while Yakuts account for 0.33% only. Sakha (Yakutia) population encompasses 126 different ethnic communities with most numerous being Yakut one (466.5 thousand or 48.7%), Russian (38%), and some indigenous ethnic groups like Evenks (2%), Evens (2%), Yukaghirs (0.6%) and other small ethnic communities comprising a total of 6%. According to current practice, most local donor registries are formed on the basis of blood banks as there already exists infrastructure for bloodborne infections assessment and there already are motivated donors in communication with blood bank ready to be recruited.

Results

Within the year 2019, Sakha (Yakutia) Laboratory Medicine Forum framework there was a round table dedicated to blood banking laboratory service improvement, in which took part a number of leading experts in the field (Godkov M. A., Shcherbo S. N., Dolgikh T. I. and others). The final decision was to create a regional bone marrow donors database. Also, this initiative found a strong support from non-profit Female Yakutia Scientists League headed by N. V. Savvina. In 2020, the initiative was supported by 2.57 million rubles from the RS(Y) administration grant used to provide equipment for preliminary HLA typing (Bio-Rad CFX-96) and a thermocycler for blood-borne infections screening in potential donors. The initiative was also promoted by volunteers (mostly medical students) on regular basis by a series of events like schools, quizzes, quests, flashmobs, lectures, etc. In 2017, the search for potential bone marrow donors for a patient named Maria S. was initiated. A total of 662 samples were collected in 3 days by a local blood bank and screened for suitable HLA alleles. Unfortunately, the compatible donor was not found at screening of this sample series. There is currently a registry of potential bone marrow donors in RS (Y), which consists of more than 100 people of different ethnicities. In August 2021, an agreement was signed with Kirov Research Institute of Hematology and Blood Transfusion. Soon, only one month after the regular HLA typing was initiated (in September 2021), the first HLA-matched donor was found for Yakutia patient. He was among the first 16 potential donors recruited, which probably was an indicator of high local registry effectiveness for RS(Y) patients.

Conclusions

Considering the complex combination of different ethnicities seen in Sakha Republic population characterized by abundance of rare HLA haplotypes not seen in most Russian donors, it is necessary to further develop a local potential hematopoietic stem cells donors Registry.

Keywords

Registry, donors, bone marrow, Republic of Sakha (Yakutia).

An improvement in blood bank infections control measures effectiveness is crucial for many organizations involved in blood banking and ones providing blood transfusions. In 2016-2020 there was a stable downward tendency in bloodborne infections’ markers positivity among blood donors. However, in 2021 the positivity rate increased from 0.76% to 0.99%. There are a lot of factors able to influence this process such as donation (primary donor or regular donation) and payment type (with or without compensation) etc. We have performed an analysis of factors able to influence bloodborne infections’ markers positivity rate.

Materials and methods

This study includes the data obtain from official statistical forms № 39 (Data on storage and processing of blood and its components) and № 64 (Data on donor blood and/or its components storage, processing, transportation and clinical use) filled in 2020-2021. The data obtained was analyzed using Student’s t-test, the confidence interval used to determine statistical significance was 5% (р ≤0.05).

Results

There was a statistically significant increase in blood and/or blood components donor rejections due to positive infections markers in 2021. These changes were evident while comparing whole rejected donor cohorts (12663 donors in 2021 vs. 9194 donors in 2020, р<0.01), as well as in subgroups of donors with positive infections markers (3616 positive for syphilis in 2020 vs. 3128 in 2020, р<0.01; 3068 positive for hepatitis B in 2021 vs. 1727 in 2020, р<0.01; 4950 positive for hepatitis C in 2021 vs. 3476 in 2020, р<0.01, and 1029 positive for HIV in 2020 vs. 863 in 2021, р=0.002). According to a number of studies the overall bloodborne infections markers positivity rate is higher among first-time blood and/or blood components donors. We have, therefore, supposed this growth to be related to higher number of first-time donors. However, the further analysis has revealed similar dynamics seen in 2021 among both first-time and regular donors for all infections markers. Neither was this dynamics caused by general donors’ number increase as proved by its detailed analysis. Rate of rejections for donors of blood and blood components due to positive infectious markers in 2016-2021 did not exceed 1-1.5% for distinct infectious markers revealed (hepatitis B, C, Syphilis, HIV infection). Although, there seems to be a correlation between total blood and/or blood components donors dynamics during the study period and number of donors rejected due to positive infections markers. This suggestion is, however, dismissed by more detailed analysis of year- to year infection testing since the incidence of positive donors also increased.

Conclusions

The documented data analysis for 2016-2021 period has demonstrated a significant increase in number of blood and/or blood components donors rejected due to positive infections markers in 2021. There was no correlation revealed, either with donation (primary donor or regular donation), or payment type (with or without compensation), or generally increased number of donors. The further detailed analysis is needed in order to determine a real cause for increase in bloodborne infections’ harboring rates.

Keywords

Blood donors, infections control, bloodborne infections.

Regular blood donors often become hematopoietic stem cell (HSC) donors as well. One of their advantages as donors is the fact that they have already passed a screening for bloodborne infections markers, most common of which are hepatitis B and C. Blood banks employ most sensitive modern diagnostic techniques including NAT and are able to screen even for early stages of infections. We performed a comparative analysis of hepatitis B and C markers frequency in various categories of blood donors, which are also candidates for HSC donor registry recruitment.

Materials and methods

Our study included 56,135 of regular FSBI RNIIHT FMBA blood donors and 16,240 first-time applicants for donation screened in 2000-2019. All of them were examined for bloodborne infections markers. The investigation extent and procedure, including diagnostic reagent specification, are regulated by Russian Federation Ministry of Health and Chief sanitary doctor’s orders. All blood donor donors were evaluated for hepatitis B, hepatitis C, HIV and syphilis markers via ELISA or immunochemiluminescent analysis using the Architect I 2000 SR automatic immunochemical analyzer. Since 2013 all blood donors has been examined for hepatitis B, hepatitis C and HIV nucleic acids presence with a help from a fully automated PCR-complex Cobas s201 system. The 2020-2021 donor survey results are not included in the analysis due to the changes introduced into donor department’s procedures during COVID-19 pandemic.

Results

Among the regular FSBI RNIIHT FMBA donors examined the viral hepatitis B markers incidence gradually decreased from 0.1% in 2000 to an average of 0.03% with no marker carriers found in some of the years. The hepatitis C markers were seen more often with 0.3% incidence in 2000 with subsequent average rate of 0.15% among subjects examined. Among the first-time donors the incidence of markers for both viruses was more significant with hepatitis B found in 1.3% in 2000 and then slightly decreasing to a rate of 0.5%, and hepatitis C markers been positive in 3.7% potential donors in 2000 and then seen in an average of 1.2% cases. From 2015 to 2019, the overall hepatitis markers positivity rate was 0.08% in regular donors and 1.4% in first-time ones, which demonstrates a slight general decrease infection frequency. In both blood donor groups the hepatitis C detection rate notably exceeds one for hepatitis B being about threefold higher.

Conclusion

Assumingly healthy people applying as candidates for donation are, nonetheless, infected in about 2% of cases (these figures are even higher in endemic regions) and are not accepted for donation due to bloodborne infections markers presence. A similar proportion of all potential first-time donors are likely to fail to become HSC Registry donors as well. In this regard, the donor social background, motivation and thorough questioning are of particular importance.

Keywords

Registry, hemopoietic stem cells, donors, bloodborne infections, hepatitis.

Among patients in need of transfusions there are people with rare erythrocyte antigens. In case of urgent transfusion there may be no compatible blood components available due to erythrocyte antigens mismatch. Also, antibodies directed against erythrocyte antigens may be a problem. There are a lot of sensitized individuals among hematological and oncohaematological patients, e.g. for those with thalassemia and sickle cell anemia it may be as high as 30%. The creation of blood donors registry containing blood typed by erythrocyte antigens may be a solution to donor/recipient pair matching problem. Since 2018 a registry of blood donors with RH, Kell, Kidd, Duffy, MNS, and Dombrock erythrocyte antigens typed functions in Russian NIIGT. We aimed to evaluate the occurrence of most clinically significant erythrocyte antigens of the RH, Kell, Kidd, Duffy, MNS, and Dombrock systems in registry donors.

Materials and methods

A total of 427 blood donors from RosNIIGT (119 women and 308 men) were examined. The median age was 35 years. All donors were typed for RH, Kell, Kidd, Duffy, MNS, and Dombrock erythrocyte antigen systems. The serological typing of RH system antigens and K antigen was performed using gel technology in ID cards (Bio Rad, USA). Alleles of genes mediating antigenic properties of erythrocytes of RH, Kell, Kidd, Duffy, MNS, Dombrock systems were determined by real-time PCR with a set of reagents RBC-FluoGene vERYfy (Inno-Train, Germany).

Results

In Kell system, the K-k+ phenotype was determined in 87.8% of donors, and the K+k+ phenotype was determined in 11.5%. The K+k-phenotype was detected only in 0.7% of donors. The Kidd system antigens were distributed as follows: Jk^a+Jk^b+ in 50.6% of subjects, Jk^a+Jk^b- in 21.3%, and Jk^a-Jk^b+ in 28.1%. In the Duffy system the Fy^a+Fy^b+ phenotype was found in 45.1% of donors, Fy^a+Fy^b in 23.0% and Fy^a+Fy^b+ in 21.9%. Fy_x antigen was present in 18 donors (4.2%) and U+var (P2) antigen was detected in 2 cases (0.5%). In the MNS system the S+s+ phenotype was detected in 43.1% of donors, S+s in 11.2% and S-s+ in 45.7%. Antigen M was present in 56.9% of the samples, antigen N in 44.3%. The Dombrock Do^a+Do^b+ phenotype was found in 47.3% of donors, Do^a+ Do^b- in 8.2% and Do^a-Do^b+ in 44.5%. Antigen C^w the RH systems was detected in 7.3%. The RH system antigens D, C, c, E, e, K serological typing results correlated with genotyping results by 98.2%. In one case the D antigen was not serologically detected and Rh affiliation was determined as negative. During genotyping an allele of the RHD gene encoding Dweak typ 4.1 was detected; Rh affiliation is positive.

Conclusions

The occurrence of RH, Kell, Kidd, Duffy, MNS, and Dombrock erythrocyte antigen phenotypes in registry donors was analyzed. Further work is planned to investigate occurrence of rare antigens types as well as registry volume expansion.

Keywords

Registry, donors, erythrocyte antigens.

Hematopoietic stem cell transplantation (HSCT) has been used in the Republic of Belarus for more than twenty years. This treatment option often presents the only chance to save the patient’s life. Effective donor search is one of the essential components of successful transplantation. The COVID-19 pandemic and further economic and social restrictions have had a significant impact on the logistics of the donor search, presenting the coordinators with a number of problems that need to be addressed immediately.

Results

The main problem is the lack of the required numbers of HLA-compatible donors in the National Registry. After 2020, the chance of finding a matched donor has significantly decreased due to the outflow of the population and the general health deterioration in the donors amid the COVID-19 pandemic. This problem is solved by searching outside the republic: Russia, Kazakhstan, Poland, Germany, and other countries. Germany and Poland remain the leading suppliers of stem cells, providing 86% of the total number of HSCTs performed in 2018-2022. This fact is explained not only by a large number of potential donors in the registries but also by the diversity of HLA phenotypes. However, the COVID-19 pandemic and the following social and economic events have significantly reduced patients’ chances for successful donor search, first of all, due to the complex logistics of this process at all stages. Thus, in 2020, the number of unrelated allo-HSCTs performed in Belarus decreased by a half (from 32 cases to 16). The current situation requires comprehensive solutions involving all interested parties (medical institutions, the Ministry of Health, services at the state border and customs, courier companies, et al.). One may list the following measures that may contribute to recovery of pre-pandemic HSCT activities: (1) visa-free regimen when entering through Minsk National Airport; (2) allowing couriers to arrive in the country without quarantine measures and providing a certificate of vaccination; (3) arrangement of delivery through third countries (Turkey, Lithuania, and others); (4) implementation of financial calculations via the intermediary banks. Looking for donors in Russia is an option. At the moment, the situation is complicated by lack of a unified Donor Registry and agreements on long-term cooperation between the institutions. However, the experience of stem cell transplantation from Russian donors suggests advances in such cooperation, by mutual engagement of the both sides. Moreover, within the framework of the Union State, it is possible to arrange effective logistics of the donorship process at all stages.

Conclusions

Current donor search is a task with many unknowns. However, HSCT remains the only chance for survival in most patients. It is our destiny to involve all potential resources, to establish partnerships, and also increase the popularity of the very idea of stem cell donation in order to timely implementation of these chances.

Keywords

Hematopoietic stem cell transplantation, donor search, registry, Belarus.

Current trends in allogeneic hematopoietic stem cell transplantation (allo-HSCT) development require mandatory immunogenetic testing (HLA-typing) of donors and recipients in order to enable the optimal donor selection for a particular patient. Therefore, the effective work of HLA laboratories is crucial to allo-HSCT success. Every year several HLA-laboratories from Russian Federation participate in international procedure of External proficiency testing (EPT) implementation for external quality control of HLA typing conducted by European Federation for Immunogenetics (EFI) since there are currently no domestic (all-Russian) external control programs. The purpose of this work is analyzing the international procedure of External Proficiency Testing (EPT) effectiveness in Russian laboratories.

Materials and methods

For the period from 2017 to 2021 a total of 10 to 16 laboratories from Russian Federation took part in annual EPT (the largest number was registered in 2019), which amounts to 25.6%-45.5% of the total number of participants.

Results

The minimal requirements for external quality control (HLA-A*, -B*, -DRB1* typing at low-resolution level) were performed only by some laboratories over a limited time period from 2017 to 2019. The majority of laboratories (57.1%-66.7%) over the past five years have presented the HLA typing results for 5 loci (HLA-A, B, C, DRB1, DQB1) at high or allelic level resolution. The HLA-DRB1 and -DQA1 loci were studied by single laboratories. About a third of laboratories (26.7%-30%) completed their external quality control program to the maximum reporting results from both low and high resolution or allelic HLA-typing. Also, 2 to 15 discrepancies were registered in laboratories while performing HLA-typing at the low resolution level (the most frequent discrepancies were revealed at the HLA-A and -C loci) except 2020, when all laboratories completed the EPT without discrepancies. The number of annual discrepancies varied from 5 to 11 in high-resolution or allelic HLA-typing (the most frequent discrepancies were revealed in HLA-DRB1 locus typing). According to the criteria of EPT assessment, 11.1%-28.6% of Russian laboratories failed to complete low resolution HLA-typing during 2017-2019. Only in 2020 and 2021 the results of all laboratories were satisfying. Successful implementation of high-resolution HLA typing by all participants from the Russian Federation was noted only once in 2018. The EPT results from 11.1% to 33.3% of laboratories were dissatisfying during remaining four years of observation.

Conclusions

The results of the analysis indicate the need of development and implementation of a national external HLA-typing quality control system and organization of continuous education for specialists of HLA-laboratories. This will increase the efficiency of laboratories, both in donor-recipient pairs selection and in hematopoietic stem cell donors registries development.

Keywords

HLA-typing, quality control, laboratory.