ISSN 1866-8836
Клеточная терапия и трансплантация

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Volume 7, Number 1
03/25/2018 06:40:54 pm
Volume 7, Number 1
Editor-in-Chief
Afanasyev B. V. (St. Petersburg, Russia)
Co-Editors-in-Chief
Wagemaker G. (Rotterdam, Netherlands)
Zander A.R. (Hamburg, Germany)
Deputy Editor
Fehse B. (Hamburg, Germany)
Managing Editor
Chukhlovin A. B. (St. Petersburg, Russia)
Editorial Board
Aleynikova O. V. (Minsk, Belarus)
Borset M. (Trondheim, Norway)
Chechetkin A. V. (St. Petersburg, Russia)
Fibbe W. (Leiden, Netherlands)
Galibin O. V. (St. Petersburg, Russia)
Hölzer D. (Frankfurt a.M., Germany)
Klimko N. N. (St. Petersburg, Russia)
Kolb H.-J. (München, Germany)
Kröger N. (Hamburg, Germany)
Kulagin A. D. (St. Petersburg, Russia)
Lange C. (Hamburg, Germany)
Mamaev N. N. (St. Petersburg, Russia)
Mikhailova N. B. (St. Petersburg, Russia)
Moiseev I. S. (St. Petersburg, Russia)
Nagler A. (Tel-Aviv, Israel)
Nemkov A. S. (St. Petersburg, Russia)
Paramonov I. V. (Kirov, Russia)
Roumiantsev A. G. (Moscow, Russia)
Savchenko V. G. (Moscow, Russia)
Smirnov A. V. (St. Petersburg, Russia)
Uss A. L. (Minsk, Belarus)
Zubarovskaya L. S. (St. Petersburg, Russia)
In this Issue
Alexey B. Chukhlovin This issue of CTT begins with a comprehensive review by Professor H.-J. Kolb concerning the role of gut microorganisms, their qualitative and quantitative alterations in the early posttransplant period and current attempts to make their composition and functions more favorable to the host organism. Another mini-review was written by Prof. Tapani Ruutu who draws attention to pharmacokinetics and optimal dosage of busulfan, an old drug which is still routinely used for cytostatic therapy in HSCT. A special survey has discovered some details of its usage in the European clinics. Posttransplant cyclophosphamide-based prophylaxis (PtCy) of acute graft-versus-host disease in leukemic patients is now widely used. However, its introduction to pediatric BMT centers needs further clinical evidence presented by Dr. Bykova et al. who have studied early and late outcomes following PtCy in young patients subjected to HCST for non-malignant diseases Myelodysplastic syndrome (MDS) is intensively studied now because its real incidence seems to be rather common with aging of human population. A meta-analysis performed by Dr. N. Tcvetkov et al. shows some correlations between distinct genetic mutations and clinical response of the MDS patients especially those treated with demethylating agents. A special study by Dr. Khamitova et al. was dedicated to parvovirus B19 which seems to be activated in the patients after cytostatic therapy. Their preliminary data still show correlations between initial parvovirus positivity and subsequent antibody response as well as association with hematological problems occurring after HSCT. The section Experimental Studies contains a review by Prof. A. Yaremenko et al. describing some current and potential applications of artificial scaffolds for craniofacial tissue engineering. Appropriate constructs are made of synthetic polymers which should be cell-friendly, non-cytotoxic and biodegradable upon their implantation. Rather practical and precise approach to sensitive HLA testing in marrow donors using monoallelic Sanger sequencing is shortly presented by Dr. E. Kuzmich et al. Another preliminary, but quite interesting, study is published by Dr. Suat Cheng Tan et al. who suggest a method of culturing adult human brain neurons from surgical samples. This approach has some prospectives and may be potentially developed for regenerative neurology. The Letter to the Editor by Dr. I. Moiseev concerns a practically important issue of individual prognosis in acute myeloid leukemia and role of modern statistics in appropriate predictions based on clinical and laboratory criteria. This is the reply to a comment by Prof. Robert P. Gale published in previous CTT issue. The letter by Moiseev is also accompanied by a response by Prof. R. Gale.

Editorial article

Editorial article
Professor Boris V.Afanasyev

Review articles

Prospectives for using artificial scaffolds in oral and craniofacial surgery: literature review
Аndrey I. Yaremenko1, Anna V. Lysenko1, Elizaveta A. Ivanova1, Alexander D. Vilesov1,2, Oleg V. Galibin1, Nikolay L. Petrov1, Pavel A. Kirillov1
1Department of Faciomaxillar Surgery, R. M. Gorbacheva Research Institute of Children Oncology, Hematology and Transplantology, Research Center; The First St. Petersburg State I. P. Pavlov Medical University, St. Petersburg, Russia
2Institute of Macromolecular Compounds Russian Academy of Sciences, St. Petersburg, Russia
Infection and GVHD
Hans-Jochem Kolb1, Daniela Weber2, Belinda Pinto Simoes3, Ernst Holler2

Clinical studies

Acute GvHD prophylaxis with posttransplant cyclophosphamide after hematopoietic stem cell transplantation (HSCT) for non-malignant disorders
Tatiana A. Bykova, Anastasia S. Borovkova, Anna A. Osipova, Varvara N. Ovechkina, Olesya V. Paina, Polina V. Kozhokar, Alexander L. Alyanskyi, Alexander D. Kulagin, Elena V. Semenova, *Boris I. Smirnov, Ludmila S. Zubarovskaya, Boris V. Afanasyev
Parvovirus B19 incidence, specific antibody response, and delayed hematopoietic recovery after allogeneic hematopoietic stem cell transplantation
Irina V. Khamitova, Irina N. Lavrentyeva, Maria Yu. Averyanova, Alexey B. Chukhlovin, Ludmila S. Zubarovskaya, Boris V. Afanasyev
Meta-analysis of studies with genome sequencing in myelodysplastic syndrome treated with hypomethylating agents
Nikolay U. Tcvetkov, Olga S. Epifanovskaya, Yulia V. Rudnitskaya, Elena V. Morozova, Ivan S. Moiseev, Boris V. Afanasyev

Experimental studies

Identification of the new HLA-B*44:02:45, DQB1*02:85, DQB1*06:210, DRB1*01:01:30 alleles by monoallelic Sanger sequencing
Elena V. Kuzmich, Alexander L. Alyanskiy, Svetlana S. Tyapushkina, Anna A. Nasredinova, Natalya E. Ivanova, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Letter to the Editor

Editorial article

Editorial article

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Professor Boris V.Afanasyev

Dear CTT authors and readers,

Immunotherapy is known to be a crucial component of systemic anticancer therapy, along with radiation and chemotherapy. It seems to be an effective means to eradicate residual tumor cells that escape conventional cytoreductive chemotherapy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a kind of adoptive immune therapy applied at increasing rates for treatment of different systemic and solid malignancies.

However, despite relative efficiency of allo-HSCT, the malignant cell resistance remains the critical issue even after this intervention. Therefore, immune therapy could be performed both before and after HSCT. A substantial number of novel treatments has emerged recently which provide a “bridge” for subsequent allo-HSCT, e.g., monoclonal antibodies against CD20 or CD30 antigens, including conjugates with cytotoxic agents. The other group includes bispecific monoclonal antibodies, like recently registered CD3/CD19 blinatumomab, which induce T cell response to the blast cells of B lineage.

Furthermore, the most promising group of novel drugs is checkpoint inhibitors, for example, anti-PD1 antibodies. Growing clinical and experimental data indicate that this group of drugs might have a role as monotherapy or in combination with other interventions in a broad spectrum of refractory malignancies. Higher efficiency of the PD-1 inhibitorsmay be achieved by optimal combining cytostatic chemotherapy and immunomodulatory drugs, e.g., lenalidomide and other agents. Their current application as monotherapy, like in Hodgkin’s disease, is not associated with high proportion of cured patients, although the initial clinical response rate is rather high. These aspects of checkpoint inhibitors make them one of the most promising “bridging” therapies, but the optimal drug dosing as well as proper timing of subsequent allo-HSCT are still to be elucidated.

The research in the field of optimal combination of novel immunotherapeutic agents and well-known approaches, including chemotherapy and stem cell transplantation, is among current priorities, both in the fundamental and clinical settings. There is definitely a large number of patients, including those with HIV infection, who will benefit from these upcoming studies.

We would be happy if our English- and Russian-speaking readers will be able to submit their original or review articles to the Cellular Therapy and Transplantation Journal. Publication in English (with extended Russian abstracts) of the data obtained by young specialists will help them to adapt for Western publication standards and to gain further experience in the research paper submission.

Review articles

Prospectives for using artificial scaffolds in oral and craniofacial surgery: literature review

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Аndrey I. Yaremenko1, Anna V. Lysenko1, Elizaveta A. Ivanova1, Alexander D. Vilesov1,2, Oleg V. Galibin1, Nikolay L. Petrov1, Pavel A. Kirillov1
1Department of Faciomaxillar Surgery, R. M. Gorbacheva Research Institute of Children Oncology, Hematology and Transplantology, Research Center; The First St. Petersburg State I. P. Pavlov Medical University, St. Petersburg, Russia
2Institute of Macromolecular Compounds Russian Academy of Sciences, St. Petersburg, Russia

Regenerative medicine is an emerging field of biotechnology that combines various aspects of medicine including cell and molecular biology, material science and bioengineering – to regenerate, repair or replace tissues. Bone regeneration is a promising approach in dentistry and is considered an ideal clinical strategy in treating diseases, injuries, and defects of the maxillofacial area. Advances in tissue engineering have resulted in the development of innovative scaffold designs, complemented by the progressmade in cell-based therapies. In vitro bone regeneration can be achieved by the combination of stem cells, scaffolds, and bioactive factors. A possible improvement in restoring damaged tissues may be achieved by loading the scaffolds with drug substances, as well as genetic material, growth factors or other proteins, promoting the tissue regeneration. This review focuses on different biomaterials currently used in dentistry, as potential scaffolds for bone regeneration when treating bone defects, or in surgical interventions, including characteristics and types of these scaffolds, and a literature review of local antibiotic delivery by combined usage of scaffolds and drug-delivery systems.

Keywords

Craniofacial surgery, scaffold, tissue engineering, stem cells, drug delivery.

Review articles

How to use busulfan in conditioning for allogeneic transplantation

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Tapani Ruutu

Clinical Research Institute, Helsinki University Hospital, Finland

Busulfan-based conditioning has been used for decades in allogeneic haematopoietic stem cell transplantation (allo-HSCT). Initially, the drug was given orally. However, variable absorption rates from the gut resulted sometimes in adverse toxic effects. Later on, intravenous administration has replaced oral administration, but many centres still use the oral route. Moreover, different centres use various administration schedules and pharmacokinetic assays to individualize busulfan dosage. A Working Party of the European Society for Blood and Marrow Transplantation (EBMT) has carried out a survey among EBMT centres about their practice in the use of busulfan for conditioning in HSCT in adults, including dosage and routes of busulfan administration, and role of pharmacokinetic monitoring. At most centres, busulfan is given intravenously, both in myeloablative and reduced-intensity conditioning. There is marked variation between centres in the details of busulfan administration. The clinical impact of this variation remains uncertain. Efforts toward a more standardized use of busulfan in the conditioning would be indicated.

Keywords

Busulfan, hematopoietic stem cell transplantation, administration route, dosage, monitoring.

Review articles

Infection and GVHD

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Hans-Jochem Kolb1, Daniela Weber2, Belinda Pinto Simoes3, Ernst Holler2

1Kolb Consulting UG, München, Germany
2Department of Hematology and Oncology, Internal Medicine III, University Medical Center, Regensburg, Germany
3Ribeirao Preto School of Medicine, Sao Paulo University, Brazil
The role of infections in allogeneic stem cell transplantation and graft-versus-host disease has gained a renewed interest because of several developments in recent years. Variable degrees of immune deficiency exist as a consequence of immune suppression until the immune system of donor origin is established; graft-versus-host disease (GVHD) and immunosuppressive treatment for prophylaxis and therapy may delay the restoration. These conditions favor infections with various microorganisms. Several improvements in prophylaxis and treatment of infections as well as reduced intensity of the conditioning regimens and improved prophylaxis of GVHD have decreased toxicity of the treatment and transplant-related deaths.
Improved antibiotics, antiviral and antifungal treatment have contributed to the greater success. However, infections with and without GVHD remain a major obstacle of allogeneic stem cell transplantation and immunotherapy. New diagnostic tools for the study of cytokines released during conditioning, the composition and alteration of the gut microbiome after transplantation and the innate immunity of the gut mucosa have given new insights into the pathophysiology of GVHD. The gut is a primary organ of T cell activation in acute GVHD; the incidence of GVHD is associated with a lower gut microbial diversity.
The composition of intestinal microbiota seems to play an important role for the pathophysiology of intestinal GvHD. Commensal bacteria, particularly Clostridiales, like Blautia, have been shown to be associated with less GvHD. The mechanism by which anaerobic bacteria suppress GVHD is still unknown, most likely due to secretion of protective metabolites like short chain fatty acids or indole and its derivatives, thus exerting antiiflammatory effects and contribute to epithelial integrity and immunological homeostasis. Modulation of intestinal microbiota composition may influence the occurrence and severity of gut GvHD.
The form of gut decontamination has also an important impact on GVHD. E.g., rifaximin is a broad-spectrum antibiotic with negligible gastrointestinal resorption that spares anaerobic bacteria and improves indoxyl sulfate production. Rifaximin preserves high microbiome diversity upon gut decontamination, as compared to ciprofloxacin and metronidazole being associated with less severe GI GvHD and improved survival.
Even kind and timely use of systemic broad-spectrum antibiotics for therapy of neutropenic infections seems to impact gut GvHD. E.g., avoidance of imipenem/cilastatin and piperacillin/tazobactam seems to improve survival by decreasing GVHD rates, probably, due to growth of Akkermannsia muciniphilia with mucus-degrading capabilities, thus, probably, promoting intestinal inflammation and GvHD. Aztreonam and cefipime, both antibiotics with anaerobic sparing effects, may be preferable. Use of antibiotics before the day of transplantation may contribute to severe intestinal dysbiosis and poor outcome of patients after ASCT.
The presence of certain strains of anaerobic bacteriae is associated with lower risks of GVHD and relapse of leukemia. Recent studies have shown that gut colonization with some strains of Blautia is associated with lesser risk of GVHD, and strains of Limus (Eubacteriaceae) is associated with a decreased relapse rate. This antileukemic mechanism is not well understood. A common finding is the production of short chain fatty acids. Hence, the question of total or selective gut decontamination is discussed controversially. Improved survival was described with the decontamination with rifaximin that is associated with surviving anaerobes and an increased production of indolsulfoxide. Treatment of GVHD of the gut has been attempted with the transfer of stool from a healthy person with some success. This may not only be the beneficial impact of bacteriae, but the composition of bacteriae with phages and other microorganisms. A recent study of the viriome found the presence of picobrna virus associated with GVHD.
The gastrointestinal mucosa is an important part of the immune system and there is a delicate equilibrium between the flora itself and the immune surveillance by the host’s immune system. There is a good evidence that the mucosal immune system plays a pivotal role in the development of the patient’s immunity against food antigens and microbial antigens thereby distinguishing between reaction and tolerance.
Viral infections are known to pave the way for subsequent fungal and bacterial infections, but complex interactions between the viruses, bacteria, fungi, nematodes and host mucosa may complicate the picture. A still largely unknown but highly important mechanism of transkingdom control may be associated with poorly studied role of phages that may modulate bacterial colonization. These interactions may be complicated by clinically applied antibiotics (absorbable and non-absorbable), antivirals and other drugs.
There are also some encouraging new ways to prevent and to treat GVHD. Moreover, one may select donors according to their immune repertoire and genetic background for T cell activation. Possibly this can be combined with an anti-leukemic efficiency based on anti-microbial activity and HLA class II DP histocompatibility. In general, the immune activation may be important that is induced by the actual microbiome and determined genetically by the donor and the host.

Keywords

Allogeneic hematopoietic cell transplantation, graft-versus-host disease, infection, prevention, gut micro

Clinical studies

Acute GvHD prophylaxis with posttransplant cyclophosphamide after hematopoietic stem cell transplantation (HSCT) for non-malignant disorders

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Tatiana A. Bykova, Anastasia S. Borovkova, Anna A. Osipova, Varvara N. Ovechkina, Olesya V. Paina, Polina V. Kozhokar, Alexander L. Alyanskyi, Alexander D. Kulagin, Elena V. Semenova, *Boris I. Smirnov, Ludmila S. Zubarovskaya, Boris V. Afanasyev

R. Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantology; Department of Hematology, Transfusiology and Transplantology, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia *The St. Petersburg State Electrotechnical University (LETI), St. Petersburg, Russia

Transplantation of allogeneic hematopoietic stem cells (allo-HSCT) is an effective treatment method for non-malignant diseases and inherited disorders. Development of acute graft-versus-host-disease (aGVHD) is a negative factor with adverse effects upon clinical outcomes. Usage of “novel” schedules for drug prophylaxis of this complication using posttransplant cyclophosphamide (PtCy) seems to decrease the GVHD risk. The aim of this study was to assess efficiency of PtCy as a tool for aGVHD prevention in the patients with non-malignant diseases of hematopoiesis and inherited syndromes.

Patients and Methods

97 patients with non-malignant blood disorders and metabolic diseases underwent allo-HSCT at the R. Gorbacheva Memorial Institute of Children Oncology and Transplantation over a period of 2005 to 2018. A total of 118 HSCTs were carried out. The aGVHD prophylaxis in 89 cases was performed by a standard schedule (with calcineurin inhibitors). 29 patients were treated according to PtCy regimen, at a dose of 50 mg/kg at days +3 and +4.

Results

Cumulative frequency of acute GVHD comprised 32%. Patients treated with PtCy exhibited lower rates of this condition compared to the group with standard prophylaxis schedule (26% vs 47%, р=0.05). Frequency of skin aGVHD was also less common in the PtCy group (23% vs 45%, р=0.046); gastrointestinal aGVHD was observed at equal rates in the both groups. Stem cell engraftment after nonmyeloablative conditioning in HSCT patients with subsequent PtCy administration proved to be sufficiently weaker compared to other patients (86 vs 50%, р=0.004). In conclusion, posttransplant GVHD prevention based on cyclophosphamide prophylaxis is an efficient method which may decrease aGVHD risk. However, one should take into account a higher non-engraftment rate as a potential hazard of HSCT when using non-myeloablative conditioning regimens and Pt-Cy-based GVHD prophylaxis.

Keywords

Allogeneic hematopoietic stem cell transplantation, non-malignant disorders, acute graft-versus-host disease, cyclophosphamide prophylaxis.

Clinical studies

Parvovirus B19 incidence, specific antibody response, and delayed hematopoietic recovery after allogeneic hematopoietic stem cell transplantation

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Irina V. Khamitova, Irina N. Lavrentyeva, Maria Yu. Averyanova, Alexey B. Chukhlovin, Ludmila S. Zubarovskaya, Boris V. Afanasyev

L. Pasteur Research Institute of Epidemiology and Microbiology, St.Petersburg, Russia; R. Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, The First St. Petersburg State Medical I. Pavlov University, St. Petersburg, Russia

Parvovirus B19 (PVB19) is a well known DNA virus which seems to be associated, e.g., with erythropoiesis disturbances. Being a latent virus, the PVB19 may become active in immunocompromised patients. However, clinical significance of PVB19 after hematopoietic stem cell transplantation (HSCT) is yet not clear. Therefore, the aim of our study was compare the PVB19 DNA levels prior to allogeneic HSCT, and at 1-2 months post-transplant, as well as search for correlations with specific antibody levels and possible effects upon hematopoietic recovery within 60 days after HSCT. Our study included 54 pediatric and adolescent patients of 0.6 to 19 years old with blood malignancies or inherited disorders who underwent allogeneic HSCT. Fifty-one patient of this group were observed for at least 60 days after HSCT. 33% of the patients were in first remission after previous treatment. Non-myeloablative conditioning treatment was used in 94% of cases. Antilymphocyte immune globulin and/or cyclophosphamide were applied as immunosuppressive therapy. Determination of the PVB19 DNA as well as herpesviruses (CMV, EBV, HSV) and polyomaviruses (BK, JC) was performed before conditioning therapy which preceded allo-HSCT, as well as on day +30 (D+30) and day+60 (D+60) post-transplant. Quantitative determination of the PV B19 DNA was performed by gene-specific real-time PCR using commercial kits. IgG and IgM antibodies to PVB19 were determined quantitatively by means of ELISA method.

Results of the study were as follows: PVB19 DNA at low levels was found in blood plasma samples of 31.5% HSCT patients. However, 68% of the patients exhibited detectable levels of IgG-anti-PVB19 antibodies (>10 IU/ ml), thus reflecting high prevalence of adaptive immune response. Generally, prevalence and mean levels of PVB19 DNA as well as concentrations of anti-PV B19 antibodies did not show any significant changes at 30 or 60 days after HSCT.

Meanwhile, a significant positive correlation was revealed between the overall PVB19 viral load and serum levels of IgG antiviral antibodies (r=0.351; p<0.0001). Moreover, positivity for PVB19 DNA by the day +30 after allo-HSCT was in all cases (14/14), associated with febrile neutropenia in the patients, thus suggesting their potential role in posttransplant infections.

Specifically, important correlations were observed between initial parvovirus DNA detection, and delayed reconstitution of erythrocytes and platelets in peripheral blood (respectively, r=-0,281; p=0.02; r=-0,303, p=0.01). Moreover, a marked correlation was shown by the day +60 between decreased neutrophils and platelet counts, and increased anti-PVD19 antibody levels. This finding may suggest an association between parvovirus activation and slower hematopoiesis recovery after allogeneic HSCT.

Keywords

Hematopoietic stem cell transplantation, parvovirus B19, activation, antiviral antibodies, myelosuppression, febrile neutropenia.

Clinical studies

Meta-analysis of studies with genome sequencing in myelodysplastic syndrome treated with hypomethylating agents

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Nikolay U. Tcvetkov, Olga S. Epifanovskaya, Yulia V. Rudnitskaya, Elena V. Morozova, Ivan S. Moiseev, Boris V. Afanasyev

R. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov State Medical University of St. Petersburg, St. Petersburg, Russia

Myelodysplastic syndrome represents a heterogenous group of clonal diseases affecting the hematopoietic stem cells underlied by different somatic gene mutations and/or altered epigenetic regulation induced by the disturbed microenvironment, as well as changes in the immune surveillance system. In many patients, the MDS is preceded by a period of non-clonal or clonal cytopenias of a non-clear significance that are determined by age-associated somatic mutations and increased leukemia risks resulting into a higher cellular proliferation, inefficient clonal growth, suppression of normal hematopoiesis, and, finally, into altered differentiation, thus causing accumulation of blast forms and a risk of evolving into acute leukemia. Substantial data on prevalence and impact of mutations on the prognosis in myelodysplastic syndrome was accessed by multiple groups however the results of several published studies are controversial. Thus we have performed an unconventional meta-analysis by accessing resulting confidence intervals both by statistical means and by creating pulled database with available individual patient data. 12 studies with 1238 patients were analyzed. The observed prevalence of mutations was the subject to significant variability (95%CI: ASXL1 13.6-29.8%; DNMT3A 7.3-12.9%; EZH2 2.4-7.0%; U2AF1 3.7-13.8%; TET2 14.2-32.5%; RUNX1 3.9-13.7%; TP53 4.7-15.2%; SRSF2 7.1-28.1%; RAS 2.2-15,1%; SF3B1 4.4-12.2%; CBL 0.1-8.9%; None, 8.0-23.3%; р<0.0001). The analysis of response to hypomethylating agents revealed improved response in patients with TP53 (95% CI 49-55%, p=0.0003), TET2(95% CI 49-52%, p=0.0001) and SRSF2 (95% CI 48-54%, p=0.0005) mutations; however the survival was worse in TP53 mutated patients (95% CI 44-49%, p=0.002) and better in SF3B1 mutated disease (95% CI 47-54%, p=0.01). The magnitude of difference was less than previously reported. The study confirmed the previous reports on the impact of TP53, TET2 and SF3B1 mutations on prognosis. Further studies on the potential prognostic markers are required, especially in patients with absence of conventional mutations.

Keywords

Myelodysplastic syndrome, sequencing, mutations, treatment response, hypomethylating agents, metaanalysis.

Experimental studies

Identification of the new HLA-B*44:02:45, DQB1*02:85, DQB1*06:210, DRB1*01:01:30 alleles by monoallelic Sanger sequencing

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Elena V. Kuzmich, Alexander L. Alyanskiy, Svetlana S. Tyapushkina, Anna A. Nasredinova, Natalya E. Ivanova, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia

Four new HLA alleles were identified using the monoallelic Sanger sequencing method: HLA-B*44:02:45, HLADQB1*02:85, HLA-DQB1*06:210, HLA-DRB1*01:01:30. A distinctive feature of the method is to implement the initial allele-specific PCR products for subsequent separate amplification of the target gene alleles. This, in turn, allows for sequencing of each allele separately and avoiding ambiguous HLA typing results observed when performing locus-specific sequencing. The isolated sequencing of specific gene alleles is a sufficient requirement for the registration of new HLA alleles, as prescribed by the World Health Organization Nomenclature Committee for Factors of the HLA System.

Keywords

Major histocompatibility complex, novel HLA alleles, monoallelic Sanger sequencing.

Experimental studies

Human brain subventricular zone-derived neural stem cells (NSCs) isolation from stroke patients – a pilot study in Hospital Universiti Sains Malaysia (Hospital USM), Malaysia

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Suat Cheng Tan a,#, In Nee Kang a,#, Abdul Rahman Izaini Ghani b, c, Shaharum Shamsuddin a

a School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
b School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
c Hospital USM, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
# Both authors contributed equally to the work described

Ischemic stroke is caused by cerebral vessel occlusion, resulting in an area of ischemia where the brain tissue irreversibly damaged due to inadequate oxygen and nutrients. Neural stem cell (NSC) grafting has emerged as a potential regenerative therapy for stroke. Here, we described a pilot clinical study of NSC isolation from subventricular zone (SVZ) of human brain. In this preliminary study, nine patients admitted to Hospital Universiti Sains Malaysia (Hospital USM) with cerebral vascular accident (CVA, i.e. ischemic or hemorrhagic stroke) and necessitating brain resection were recruited. During surgery, SVZ biopsies were excised, weighted and the morphological structure of clinical biopsies were recorded. Then the biopsies were digested and cultured using neurosphere or monolayer culture methods. Out of nine clinical samples, we successfully obtained neurospheres-like cells from five of the patients’ SVZ biopsy, while monolayer culture grew out from two patients’ SVZ biopsy. Characterization assay using immunocytochemistry probed with nestin (NSC marker) successfully proved that these cells were NSCs. For the rest of samples, the isolation of SVZ derived-NSC was unsuccessful due to the insufficient SVZ tissue sampling. The tissue obtained were mainly connective or blood tissue, proved by the accumulation of floating cells after 24 hours in culture. In conclusion, NSCs were successfully obtained from some stroke patients SVZ. However, the quantity and quality of isolated cells are tissue-dependent, thus, surgical sampling technique and biopsy sizes are important in ensuring a good NSC isolation efficiency. This study could be an essential guidance for future NSC isolation study in our department.

Keywords

Primary neural stem cell isolation, stroke therapy, Hospital Universiti Sains Malaysia (Hospital USM), neurosphere, monolayer culture.

Letter to the Editor

Transplants for аcute myeloid leukaemia in 1st remission: standard of care or something else?

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Keywords

Acute myeloid leukemia, clinical outcomes, prognosis, statistics.