Immunology Center Research Articles

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort

Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Hematopoietic stem cell transplantation with TCRαβ+/CD19+ - graft depletion for hemophagocytic lymphohistiocytosis

The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. We present the outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) with TCRab+/CD19+ graft depletion in patients with genetic hemophagocytic lymphohistiocytosis (HLH) at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology from 2012 to 2019. Thirty-six patients with various HLH: familial HLH (n = 20), X-linked lymphoproliferative disease (XLP) type 1 (n = 4), XLP type 2 (n = 9), Griscelli syndrome (n = 1), Chediak–Higashi syndrome (n = 2) received HSCT. Conditioning regimens were based on treosulfan in 9 patients, or on two alkylating agents: treosulfan with either melphalan, or thiotepa in 27 patients; all 36 patients received fludarabine and serotherapy. Thirty-two patients received rituximab 100 mg/m2 the day before stem cell infusion. Post- HSCT “graft versus host” disease (GvHD) prophylaxis was used in 29 patients. As a graft source peripheral blood stem cells from matched unrelated (n = 23), matched related (n = 3) and haploidentical family (n = 10) donors after TCRαβ+/CD19+ graft depletion were used. The cumulative incidence of primary and secondary graft failure in all patients was 0.11 (95% CI 0.04–0.29). The incidence of acute GvHD was limited to stage I-II. Overall survival was 0.91 (95% CI 0.82–1) without any significant differences in various donor groups (p = 0.33) as well as different conditioning regimens (p = 0.75). Allogeneic HSCT with TCRαβ+/CD19+ graft depletion after treosulfan-based conditioning is effective and safe technology for patients with genetic HLH.

Allogeneic transplantation of hematopoetic stem cells in acute leukemia in children, adolescents and young adults in the Republic of Belarus

Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized method for treating children with a very high risk group for acute lymphoblastic leukemia (ALL) and a high risk group for acute myeloid leukemia (AML). The use of allogeneic HSCT for certain risk groups of acute leukemia significantly improves the survival of these patients compared to chemotherapeutic regimens. The aim of this study was to identify the causes of failure of HSC transplantation in children with acute leukemia in a homogeneous group of patients and the possibility of further improvement in survival rates. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (Republic of Belarus). The study included 101 patients with ALL and 65 patients with AML who underwent the first HSCT, in accordance with the first-line treatment protocol or relapse for 2 consecutive time periods (1998–2008 and 2009–2018). For the entire group of patients, an increase in overall (by 13%) and event-free survival (by 7%) was revealed due to a decrease in post-transplant mortality not related to relapse by 16% (p = 0.077). Significant improvement in survival over time occurred in the group of patients with acute or chronic “graft versus host” disease. The data obtained indicate that all patients with acute leukemia who have indications for HSCT in the first line of treatment or relapse should be transplanted from any available donor, as this will significantly increase their chances of recovery.

Development of flow cytometry assay for Wiskott–Aldrich syndrome diagnosis by WASP protein evaluation

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microplatelet thrombocytopenia, eczema, frequent infections and an increased risk of autoimmune disorders and malignant neoplasms. Mutation detection in WAS gene is the gold standard for diagnosis of this disorder. This gene encodes a WASP protein, which works as regulator of cell cytoskeleton and is involved in the transmission of many intracellular signals. Nowadays there is no rapid and reliable method that allows to confirm WAS in a short period of time. Early detection of WAS in patients enables initiation of a donor search and preparation for the HSCT procedure. It also helps to avoid the development of severe and life-threatening conditions during waiting for genetic confirmation of the diagnosis by using pathogenetic therapy. Currently flow cytometry is one of the leading laboratory methods that permits to get the information about the expression of a protein in several hours. The study below describes rapid and reliable based on flow cytometry assay for WAS diagnosis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study included 46 patients with suspected WAS from 2 months to 17 years old. Patients were examined from January 2018 to January 2020. WAS gene defect was confirmed in 35 patients. It was calculated that normal threshold value for WASP expression is 7.07 with sensitivity and specificity 100% and 93.1% respectively. Besides negative correlation between WASP expression index and WAS clinical severity was shown (r = –0.63). This flow cytometry assay can be used for chimerism detection in WAS patients after HSCT. The flow cytometry assay for WASP protein evaluation is rapid, highly sensitive and highly specific. It allows to speed up diagnosis of this disorder.

The effect of the minimum residual disease on the risk of relapse at allogeneic hematopoietic stem cell transplantation in children, adolescents, and young adults with acute lymphoblastic leukemia

Minimal residual disease (MRD) is an independent predictor of relapse risk for childhood acute lymphoblastic leukemia(ALL). The aim of study to investigate impact MRD by real-time quantitative polymerase chain reaction before (day –21) andat +30 ± 10, +60 ± 10, +100 ± 10, +180 ± 10, +365 ± 10 days after hematopoietic stem cell transplantation (HSCT), and PCR-chimerismon transplant outcomes children with ALL. The study was approved by the Independent Ethics Committee and the Scientific Councilof the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (Republic of Belarus). Fifty one patientswith ALL underwent allogeneic transplantation in remission (period 12.2010–12.2017, median follow-up 2,8 years). 3-years eventfreesurvival (EFS) and cumulative incidence of relapse (CIR) were 71.6 ± 17.1% and 14.3 ± 14.3% respectively for patients (n = 7)with pre-transplant MRD < 10-4 and 0% (n = 4, p = 0.0046) and 50.0 ± 29.2% (p = 0.3111) respectively for MRD ≥ 10-4. After HSCT(n = 29) 3-years EFS and CIR were 22.2 ± 13.9% and 66.7 ± 18.1% respectively for recipients (n = 9) with MRD ≥ 10-4 at leastin one analyzed point. In comparison, patients with MRD < 10-4 at all points (n = 20) had EFS and CIR 70.0 ± 10.2% (p = 0.0172)(HR = 12.3; 95% CI: 2.33–64.87; p = 0.0031), and 5.0±5.0% (p = 0.0004) (HR = 50.7; 2.5–97.5% CI: 1.60–1608.56; p = 0.0260)respectively. Patients with mixed chimerism at least in one analyzed point since day +30 to +365 hadn't significant differences OS,EFS, CIR but were worse (57.1%, 40.0% and 50.0% respectively) in comparison full chimerism patients (79.5% (p = 0.248), 71.4%(p = 0.072) and 20.0% (p = 0.070) respectively). MRD is significant predictor of relapse risk for childhood ALL at time of HSCT.MRD < 10-4 patients have significantly better EFS and CIR in comparison MRD ≥ 10-4 patients before and after HSCT.

Muscle strength and vegetative support at childhood on different stages of hematopoietic

Conditioning for hematopoietic stem cells transplantation (HSCT) and long isolation period often results in movement disorders to patient undergoing HSCT. An increesing number of reaserch to showing of the importance physical therapy at different stages of HCST. Objective. Сomparative assessment muscles strength and vegetative support childhood at different stages of HSCT for the determine timing for the motor rehabilitation. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. In the prospective comparative not randomized clinical trail were enrolled 27 patients aged 6–14 (11) years with acute lymphoblastic leukemia (ALL) (n = 8), acute myeloblastic leukemia (AML) (n = 8), sever aplastic anemia (n = 2) and primary immune deficiency (n = 9) at different stages of HSCT therapy. For all patients muscle strength and hemodynamic level in orthoclinistatic test were conducted before HSCT (-5 days of conditioning), after HSCT (+5 days after transplantation), and upon leaving hospital units (+30 days after HSCT). On the conditioning stage patient at ALL and AML diagnosis group had hyperchronotrope and hypostenic vegetative reaction type, while normostenic and normochronotrope vegetative reaction type at not malignant diseases diagnosis group. Muscle strength score at ALL and AML diagnosis group average was between 3–4 score, and 4–5 score at not malignant diseases diagnosis group. On the +5 days after HSCT in all researched diagnosis groups at 100% cases experienced hypostenic and hyper chronotrope vegetative type of reaction, and reduced muscle strength average 2 score. On the +30 days after HSCT at ALL diagnosis group experienced hypochronotrope and hypostenic type of vegetative reaction at 75% and 87.5% respectively, at AML diagnosis group experienced hypochronotrope and hypostenic type of vegetative reaction at 87.5% and 75% respectively, and at not malignant diagnosis group experienced hyperchronotrope and hyperstenic type of vegetative reaction at 81,8% cases. In the remaning cases at all diagnosis group experienced normostenic and normochronotrope type of vegetative reaction. Average muscle strength score at ALL and AML diagnosis group was 4 score. Average muscle strength score at not malignant diagnosis group was between 4–5 score. It’s important to get an early start physical therapist to childhood receiving of HSCT therapy.

Apheresis of peripheral blood stem cells in children with very low body weight: a single centre experience

Polychemotherapy, accompanied by autologous hematopoietic stem cell transplantation, can improve the results of long-term survival of patients with cancer and some non-cancer diseases. Mobilizing and collecting hematopoietic stem cells in children with very low body weight can be a difficult task. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. 19 children with extremely low body weight was included in the current study. The median age was 8 (5–14) months, the median of body weight 7.5 (5.8–8.8) kg. Apheresis was performed in an ICU, using sedative therapy and in compliance with the conditions for the prevention of anemia, hypovolemia, hypothermia. 19 hematopoietic stem cell apheresis were performed using the Spectra Optia MNC separator program. Mobilization of CD34+ cells was performed with filgrastim; three children were additionally given plerixaphor. All 19 hematopoietic stem cell apheresis were successful: the median of collected CD34+ cells was 18.7 × 106/kg (8.6– 60.6 × 106/kg), the median apheresis duration was 204 (161–351) min. Serious side effects during apheresis were not recorded, however, in 6 children (31%) we encountered difficulties in the process of installing central venous access. The collection of hematopoietic stem cells for the future high-dose chemotherapy with autologous hematopoietic stem cells is a feasible task even for very young children with extremely low body weight. Correct preparation for manipulation, taking into account all possible risk factors and technical features, can avoid serious complications.

SARS-CoV-2 and HIV coinfection: clinical experience from Rhode Island, United States.

IntroductionSevere Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) has infected >6 million people worldwide since December 2019. Global reports of HIV/SARS‐CoV‐2 coinfection are limited. To better understand the impact of the coronavirus disease 2019 (COVID‐19) pandemic on persons with HIV and improve their care, we present an outpatient and inpatient clinical experience of HIV/SARS‐CoV‐2 coinfection from Rhode Island, US.MethodsWe describe outpatient and inpatient preparedness for the COVID‐19 pandemic, and present a case series of all known patients with HIV/SARS‐CoV‐2 coinfection at The Miriam Hospital and Rhode Island Hospital, and The Miriam Hospital Infectious Diseases and Immunology Center, in Providence, Rhode Island, US.Results and discussionThe Infectious Diseases and Immunology Center rapidly prepared for outpatient and inpatient care of persons with HIV and SARS‐CoV‐2. Between 30 March and 20 May 2020, 27 patients with HIV were diagnosed with SARS‐CoV‐2. Twenty were male, six female and one transgender female; average age was 49 years; 13/27 were Hispanic and 6/27 were African American. All had HIV viral load <200 copies/mL and were on antiretroviral therapy with CD4 count range 87 to 1441 cells/µL. Twenty‐six of the 27 had common COVID‐19 symptoms for one to twenty‐eight days and most had other co‐morbidities and/or risk factors. Nine of the 27 were hospitalized for one to thirteen days; of those, three lived in a nursing home, six received remdesivir through a clinical trial or emergency use authorization and tolerated it well; eight recovered and one died. Overall, 17% of known Center people had HIV/SARS‐CoV‐2 coinfection, whereas the comparable state‐wide prevalence was 9%.ConclusionsWe highlight challenges of outpatient and inpatient HIV care in the setting of the COVID‐19 pandemic and present the largest detailed case series to date from the United States on HIV/SARS‐CoV‐2 coinfection, adding to limited global reports. The aggregated clinical findings suggest that the clinical presentation and outcomes of COVID‐19 appear consistent with those without HIV. Whether SARS‐CoV‐2 infection is more frequent among persons with HIV remains to be determined. More data are needed as we develop our understanding of how HIV and antiretroviral therapy are affected by or have an impact on this pandemic.

Observation of granulocyte function during ex vivo thrombus formation for patients with ANKRD26-associated thrombocytopenia

ANKRD26-associated thrombocytopenia is a non-syndromic hereditary thrombocytopenia for which there are currently no formal diagnostic criteria. It is known that the probability of myeloid leukemia in patients with pathogenetic variants in the ANKRD26 gene significantly increases, however, studies of the functioning of granulocytes in this pathology have not been conducted. Aims: Analysis of the functioning of granulocytes and platelets during ex vivo thrombosis in patients with ANKRD26-associated thrombocytopenia. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Two patients and 10 healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed by continuous flow cytometry. Ex vivo thrombus formation and granulocyte functioning were observed on a fluorescence microscope in parallel-plane flow chambers containing fibrillar collagen. Upon physiological activation (ADP, collagen) of patients’ platelets in vitro, there were no significant differences between the platelets of patients and healthy donors. However, the observed ex vivo size of platelet aggregates was significantly reduced in comparison with healthy donors and published data on patients with other thrombocytopenias. The observed number and activity (movement velocity) of granulocytes of patients was within normal values. However, significant morphological differences were observed for granulocytes of patients compared with granulocytes of healthy donors: there was an increased spreading of granulocytes, in particular, expressed in a large number of thin pseudopodia, as well as an increased curvature of the motion trajectories of granulocytes. Ex vivo observation of thrombus formation in patients with ANKRD26- associated thrombocytopenia, a significantly reduced thrombus size is observed with normal platelet activity and increased variability in the shape of granulocytes.

Specific features of intracellular calcium signalling, distinctive for Wiskott-Aldrich syndrome patients

Wiskott–Aldrich syndrome (WAS) is a rare X-linked immunodeficient disease accompanied by microthrombocytopenia, which leads to spontaneous/post-traumatic haemorrhages. It has been demonstrated that WAS is caused by gene mutation of WASP protein, which is participating in the processes of actin polarization and actin cytoskeleton re-organisation. It is yet unknown how this mutation affects intracellular signalling and functional responses of platelets of patients with WAS. Assessment of the intracellular calcium signalling, shape change and fibrinogen binging by the platelets of WAS patients. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Three patients with WAS and three healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed on a BD Facs Canto II flow cytometer. To measure the calcium concentration in the platelet cytosol, the Fura-Red fluorophore was used, platelet shape change upon activation was evaluated by side scattering of cells at a wavelength of 488 nm, platelet integrin activation was evaluated by binding of fluorescently-labeled fibrinogen. During activation, the platelet concentration was 1000 cells per ul to avoid the effects of secondary activation. In quescent state of platelets, an increased concentration of calcium in the cytosol of platelets of patients was observed compared with platelets of healthy donors. In response to stimulation, the highest achievable calcium concentrations were comparable in both cases. The binding of fibrinogen to platelets in patients was not significantly changed compared to healthy donors. On the other hand, the change in the shape of the cells in response to activation, expressed as a percentage, was more significant in patients than the change in the shape of the platelets of healthy donors. With similar maximum responses to stimulation by all agonists, the concentration of calcium in resting platelets, as well as the change in the platelet shape of patients with WAS is significantly higher than that of healthy platelet donors. These results can be explained by the increased ratio of the platelet membrane area to their volume.

Use of romiplostim for newly diagnosed immune thrombocytopenia in children

Immune thrombocytopenia (ITP) is a disease with a heterogeneous clinical manifestation. In the majority of children newly diagnosed ITP is a self-limited benign disorder, while chronic ITP develops rarely. The clinical onset of ITP can occur in very different ways: from nearly invisible skin hemorrhage to severe life-threatening bleeding. Conventional treatments promote a response in most patients, but in a small number of children thrombocytopenia is unresponsive. In this article, we describe our experience of the clinical use of romiplostim in children with severe unresponsive newly diagnosed ITP. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The severity of bleeding decreased significantly after the start of romiplostim therapy in all cases. Durable complete (platelets &gt; 100 × 109 /l) response was achieved in five out of six patients 4 to 8 weeks after starting therapy. Three children have remained in lasting remission for 1 to 3 years after the discontinuation of romiplostim. There were no adverse events associated with romiplostim.

The results of treatment in children with AML and initial hyperleucocytosis according to the AML-MM-2006 Protocol

The prognostic role of HL in AML in children is a matter of a discussion. 185 patients were treated for AML in our center, 36 of 185 had HL (19.5%). The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Median Le was 97 × 109 /l (50–428 ± 109 /l). Standard risk group were 4 pts, intermediate – 8, hight – 24. The most common variants were M4/M5 in FAB classification – 30 pts and genetic rearrangement – MLL in 15 of 36 pts. Thirty-five patients with HL received cytoreduction course and ADE. After that, all patients received ADE and 21 pts second part of induction – course HAM. Remission was achieved in 27 (75%) out of 36 pts. HSCT was performed in 23 pts. Thirteen out of 36 patients with HL died: 4 (30%) – due to leukostasis complications. OS for HL group was 0.56 ± 0.09, for non-HL group was 0.75 ± 0.04, p = 0.005; EFS (HL) 0.42 ± 0.09, EFS (non-HL) 0.49 ± 0.04, p = 0.026. Also, differences in I CR achievement, median of remission length and death before remission between two groups were statistically significant (p = 0.036; p = 0.028; p = 0.021 respectively). OS and EFS in patients with M4/M5 with HL &gt; 50 ± 109 /l were better than in patients all FAB with HL &gt; 100 ± 109 /l, OS 0.71 ± 0.1 vs OS 0.43 ± 0.1 (p = 0.012); EFS 0.54 ± 0.1 vs EFS 0.29 ± 0.1 (p = 0.038) respectively. HL significantly worsens OS and EFS in children with AML.

Clinical and laboratory characteristics of clozapine-treated patients with schizophrenia referred to a national immunodeficiency clinic reveals a B-cell signature resembling common variable immunodeficiency (CVID)

AimsAn association between antibody deficiency and clozapine use in individuals with schizophrenia has recently been reported. We hypothesised that if clozapine-associated hypogammaglobulinaemia was clinically relevant this would manifest in referral...

Treatment of relapse/progression of the disease in patients with neuroblastoma in the Republic of Belarus over a 20-year period: a cohort study

Introduction. Neuroblastoma (NB) — is a malignant tumor of sympathetic nervous system in children. In our country there are about 50 % of patients in high risk group, 50 % have a relapse or a progression of the disease which is the main reason for their death. The purpose of the study . Our aim was to characterize a relapse/progression of NB in children in the Republic of Belarus, the analysis of results of treatment of patients from 1997 till 2017. Materials and methods . From October, 1997 to December, 2017 in Republican Scientific and Practical Center for Pediatric Oncology, Hematology and Immunology 293 protocol and observed patients received 1st line therapy. Out of them 73 patients had a progression or a relapse of the disease. This group of patients was taken for the analysis. Results. 14-year overal survival of patients with a relapse/progression of the disease was 21 + 6 % (n = 73), event free survival 18 + 5 % (n = 53). Patients older than 12 months at the time of diagnosis were statistically significantly more likely to recur, with 4th stage disease and having N-MYC amplification, diploid karyotype in the tumor. Adverse relapse options for localization are about 65 % (generalized — 41 %, osteomedullary — 12.3 %, relapses with CNS damage — 12.3 %). The most curable variants of relapses are found in 35 % (n = 25) of cases. In patients from the group of favorable and intermediate prognosis, relapses in the zone of primary tumor localization were significantly more frequent (p < 0.0001), bone loss in the relapse of the disease was significantly less common (p < 0.0001). The best results with the use of standard therapies are relapse with the primary tumor zone and soft tissue relapse (by 5 year relapse-free survival — 25% and 36 % respectively). Treatment of osteomedullary and generalized relapses using the methods available to us is not effective (EFS is 0). Conclusion. Further detailed study of the biology of the tumor is necessary, in order to create for the therapy of the disease targeted drugs and other biologically based approaches, such various types of immunotherapy.

Investigation of a candidemia outbreak caused by C. parapsilosis in a pediatric hematology/oncology center

Objective. To investigate a candidemia outbreak caused by C. parapsilosis in a clinical unit of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology (NMRC PHOI). Materials and Methods. A total of 35 isolates of C. parapsilosis obtained from clinically significant specimens and swabs, including hands of nursing staff of the NMRS PHOI, over the 2018-2020 were genotyped in this retrospective study. Identification of C. parapsilosis isolates was performed by microbiological methods. The clonal structure of C. parapsilosis isolates was investigated by polymerase chain reaction followed by fragment analysis of microsatellite repeats (short tandem repeats, STR markers). Results. The results of the study showed genetic diversity of the population of C. parapsilosis isolates over the 2018–2020 in the NMRC PHOI. A total of 27 genotypes were identified, one of which caused candidemia in 6 patients. Conclusions. The study results confirmed the nosocomial candidemia outbreak and showed the fragment analysis of STR-markers may be used for epidemiological investigations of outbreaks in hospital settings.

Therapy intensification in high-risk neuroblastoma patients with poor response to standard induction: experience of Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology

High-risk neuroblastoma (NB) is characterized by unsatisfactory treatment results and low probability of long-term survival despite the multimodal therapeutic approach (chemotherapy, surgical treatment, radiation therapy, autologous hematopoietic stem cell transplantation, etc.). One of the prognostic factors in this cohort of patients is the response to induction therapy. The article presents the experience of the intensification of induction therapy in 12 patients with high-risk NB with a poor response (mixed response, stable disease) to standard induction therapy who received treatment at Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, assessing its impact on the prognosis of the disease. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Patients received an additional two courses of chemotherapy with the inclusion of a type I topoisomerase inhibitor topotecan (TCE – topotecan, cyclophosphamide, etoposide). This regimen of intensification of therapy has demonstrated its feasibility. The main grade 3–4 toxicity was hematologic. An improvement in response was achieved in 5/12 (41.6%) patients. However, long-term results of therapy remained unsatisfactory. The 3-year EFS was 16.7% (95% CI 0.0–37.8), the 3-year OS was 50.0% (95% CI 21.7–78.3). Thus, the intensification of therapy in patients with high-risk NB with a poor response to standard induction therapy did not improve treatment outcomes.

Hepatoblastoma relapses with a normal level of alpha-fetoprotein: report of two cases

Hepatoblastoma (HB) is the most common malignant liver tumor in children. The level of alpha-fetoprotein (AFP) is used for monitoring the response to antitumor therapy and for diagnosing relapses. The occurrence of HB relapses with normal AFP levels in patients who had elevated levels of this tumor marker at disease onset is considered to be an uncommon situation. The aim of this study was to describe cases in which AFP-negative hepatoblastoma relapses developed. The study participants included two HB patients who were initially stratified into high-risk and standard-risk groups and who received treatment at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and the Petrovsky National Research Center of Surgery inMoscow. At the moment of relapse acknowledgement these patients had normal AFP levels, which was confirmed by serial measurements. Immunohistochemistry (IHC) tests were performed on paired samples of the tumor at disease onset and at disease relapse respectively to evaluate AFP expression. The presented clinical cases demonstrate that normal AFP levels, when accompanied by suspicious clinical symptoms, do not allow to exclude an HB relapse. The possibility of relapse with a normal AFP level reinforces the importance of following the current plan of screening tests, which includes not only an evaluation of AFP levels, but also other tests such as a chest X-ray and an abdominal ultrasound. Parents gave their consent to use information about the child in the article.

Comparison of total body irradiation using TomoTherapy and volume-modulated rotational radiation therapy Elekta. A single center experience on pediatric patients

This study presents the experience of one center in the development and implementation of two different methods of totalbody irradiation (ТОТ) based on radiation therapy with intensity modulation, namely, Helical TomoTherapy (HT) and Volumetric Modulated Arc Therapy (VMAT), implemented on the classic linear accelerator Elekta Synergy. The aim of the work is the comparison of two TBI methods, as well as a description of the process of introducing these methods into the routine practice of the radiotherapy department. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev Nationa Medical Research Center of Pediatric Hematology, Oncology and Immunology. Prescribed total doses: PTV – 12.0 Gy with a single fraction of 2.0 Gy twice per a day, at least 95% of PTV should have received a dose of 11.4 Gy, the volume of each lung receiving 8.0 Gy should not exceed 40%, the average dose in each of the kidneys should not exceed 9.0 Gy, the dose in the anterior segment of the eye should be reduced as much as possible while maintaining the dose in surrounding part of PTV. In total, from July 2014 to December 2018, 156 patients received TBI, 128 of which on TomoTherapy and 28 on Elekta. The use of a standardized approach to TBI from July 2015 to December 2018 made it possible to introduce TBI into the routine practice of the radiotherapy department. A standardized approach to TBI was implemented with the irradiation of 17 on Elekta and 91 on TomoTherapy. It allowed us toobtain uniform reproducible results in terms of dosimetric criteria. The volume of lungs receiving a dose of 8.0 Gy did not exceed 40% for TomoTherapy and for Elekta, the average dose in the kidneys did not exceed 8.0 Gy for both modalities. In the standardized versions of TBI, 10.0 Gy cover at least 95% of the ribs volume, in accordance with the prescription. In both cases, the quality of PTV coverage met the prescribed criteria, namely, at least 95 percent of the volume of PTV received at least 95% of the prescribed dose (11.4 Gy). The minimum dose in the lungs was 6.2 ± 0.2 Gy and 5.1 ± 0.4 Gy, for TomoTherapy and Elekta respectively. The presence or absence of the clinical significance of this criterion is to be assessed. The developed TBI methods allow to deliver the prescribed dose to the target while maintaining the specified dose levels in the organs at risk. However, TomoTherapy plans have better target coverage and homogeneity than VMAT-based plans for Elekta. The use of a standardized approach makes it possible to quantify treatment plans for these techniques.

Clinical and molecular characteristics of WNT medulloblastomas

WNTMBaccounts for about 10% of all medulloblastomas and have a favorable outcome in patients under 16 years according international data. We analyzed clinical and molecular characteristics of 20 patients with WNTMBpatients. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The molecular group verification was performed by Nanostring gene expression profiling. The detection ofCTNNB1andTP53gene mutations was carried out with Sanger sequencing. WNT Medulloblastoma are mainly classic (18/20 patients), non-metastatic (19/20 patients). Gross total resection was performed in 18 patients. SomaticCTNNB1mutations were found in 17 patients, somaticTP53were identified in 2 patients. 19/20 assessable patients are disease-free with a median follow-up of 23 months. One patient died from the progression of the disease. WNT MB patients have an overall favorable outcome, even for metastatic andTP53positive tumors. The reduction in the intensity of therapy is indicated forWNTMB in case of reliable identification ofWNTMB.

2548. Provider Adherence to Cervical Cancer Screening in HIV Patient Populations

BackgroundAntiretroviral therapy has nearly normalized the life expectancy of people living with HIV (PLWH). However, malignancies still remain a major cause of morbidity and mortality in PLWH, and thus an important part of the clinical visit is age appropriate screening and referral to care, from Lancet 2019 for the poster. Most cases of cervical cancer occur in women who were either never screened or were screened inadequately. Over-screening for cervical cancer, on the other hand, leads to unnecessary stress and procedures, adding increased costs to the patient and to the healthcare system. The central aim of this project is to evaluate provider adherence at the Immunology Center (IC) to cervical cancer screening guidelines and to identify factors associated with over and underscreening.MethodsA retrospective chart review from January 1, 2015, to December 31, 2017 was performed. Study included HIV-seropositive women seen at the IC between April 2014 and June 2018. Exclusion criteria includes prior hysterectomy, abnormal cytology, cervical excision procedures, or other causes of immunosuppression.ResultsOf the 803 HIV-positive women identified, n = 262 met criteria for inclusion in the study. Overall adherence was 48%, with statistical significance found in cervical cancer screening between MDs and NPs, with an OR = 2.51 (P < 0.01). In regard to gender of provider, statistical significance in over screening was found between male and female providers, with an OR = 4.3 (P < 0.01), and in under screening between male and female providers, with an OR = 0.43 (P < 0.05). Over screening led to 44 excess pap smears over a 2-year period, yielding an excess cost of $6461. HPV co-testing was underutilized, with only one-third of encounters having HPV testing performed.ConclusionThis project gives us the opportunity to reeducate and retrain the clinical staff and practitioners providing cervical cancer screening at the Immunology Center. This is an ongoing quality improvement project, where adherence will be reassessed on a continuous basis at one-year intervals to ensure compliance with guidelines-based cervical cancer screening among female HIV seropositive patients at the Immunology Center. Disclosures All authors: No reported disclosures.