Download Title page Immunological reconstitution in children after completing

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Title page
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Immunological reconstitution in children after completing conventional chemotherapy of
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acute lymphoblastic leukaemia is marked by impaired B cell compartment
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Koskenvuo Minna, MD, PhD1, 4, Ilse Ekman, PhD student5, Emmi Saha, MD student1, Salokannel
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Ellinoora, MD1, Jaakko Matomäki, PhD2, Jorma Ilonen, MD, PhD5, Kainulainen Leena, MD, PhD1,
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Arola Mikko, MD, PhD3, Lähteenmäki Päivi Maria, MD, PhD1.
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1. Department of Pediatrics, Division of Pediatric Hematology and Oncology, University of
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Turku and Turku University Hospital, Turku, Finland
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2. Clinical Research Centre, Turku University Hospital
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3. Department of Pediatrics, Division of Pediatric Hematology and Oncology, Tampere
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University Hospital, Tampere, Finland
4. Department of Pediatrics, Division of Pediatric Hematology and Oncology and Stem Cell
Transplantatio, Children’s Hospital, University of Helsinki, Helsinki, Finland
5. Immunogenetics Laboratory, University of Turku, Turku, Finland
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Corresponding author:
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Minna Koskenvuo, MD, PhD
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Division of Pediatric Hematology and Oncology and Stem Cell Transplantation
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Children’s Hospital, University of Helsinki
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Stenbäckinkatu 11, 00290 Helsinki, Finland
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phone: +358 9 471 72720, gsm: +358 50 427 0424, fax: +358 9 471 74707
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Email [email protected]
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Word counts: Abstract 90, main text 1261
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Tables and figures: 2 figures, 1 supplemental figure, 1 supplemental table
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Key words: leukemia, chemotherapy, immune recovery, B cell, T cell
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Running title: Immune recovery of children with standard and intermediate risk ALL.
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Abstract
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Humoral and cellular immunity were studied in 28 children completing conventional treatment of
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standard or intermediate risk ALL. Both naïve and memory B cells were most severely affected and
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showed slow recovery during the two-year follow-up while T cell compartment showed only minor
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changes. Immunoglobulins and their subclasses, complements and antibodies against vaccine-
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preventable diseases were not significantly affected. In conclusion, immune recovery after
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conventional chemotherapy for standard and intermediate risk acute lymphoblastic leukemia is
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marked by B cell depletion but otherwise did not show any severe deficiencies in lymphocyte
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function.
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Introduction
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The consequences of currently used pediatric ALL treatment protocols on immune recovery are
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largely unknown. There are reports of reduction in naïve T cells while the memory compartment
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survives relatively well resulting in only mild defect in overall number of T cells 1-4. The data
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on B-cell recovery are sparse. Previous studies have shown considerable B cell decline both in
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bone marrow and peripheral blood followed by rapid recovery after cessation of chemotherapy
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5-6. On the other hand, there are reports suggesting long-term impairment of particular B-cell
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subpopulations leading to significant defects in B-cell function and humoral immunity even
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years after the ALL treatment 7.
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The aim of our two-year prospective study was to evaluate the rate and quality of the
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immunological recovery of children after cessation of conventional treatment for standard and
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intermediate risk ALL.
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Results
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Patients
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Between November 2008 and August 2011 28 children (mean age 8.5 years, range 4.3 – 19.2
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years) with precursor-B-ALL treated according to the NOPHO ALL-2000 and NOPHO ALL-
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2008 protocols at two centres in Finland, Turku and Tampere University Hospitals, were
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prospectively analysed for their immune recovery after completing the conventional
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chemotherapy for standard (SR) and intermediate risk (IR) ALL. The immunological status was
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evaluated at the time of cessation of chemotherapy and 3-monthly during the first year and 6-
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monthy during the second year post-ALL treatment.The study was approved by the Ethics
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Committee of Turku University Hospital. Informed consent was obtained from all patients and
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their guardians.
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Evaluation of cellular immunity
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None of the patients had severe neutropenia (neut < 0.5 10E9/l), and the median lymphocyte
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counts stayed within normal range (median 1.14 x 10E9/l in SR and 1.12 in IR) during the whole
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two-year follow-up, although some children in both risk groups had lymphopenia in single time-
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points during the first year of the follow-up (Supplemental Table I).
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Analysis of lymphocytes was performed using conjugated monoclonal antibodies (BioLegend,
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San Diego, CA) specific for CD4, CD8, CD45RA, CD25, CD27, CCR7, CD19 and CD127 cells.
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The absolute cell counts of lymphocyte subpopulations are presented in the Figure 1 and 2 and in
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the supplemental Figure 3. Due to lymphopenia and due to prospective approach of the study we
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could not analyse all the samples per time-points. The number of the patients analysed per time-
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point were 18/28 (0 m), 23/28 (3 m), 24/28 (6 m), 23/28 (9 m), 22/28 (12 m), 19/28 (18 m) and
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14/28 (24 m). At the time of the cessation of leukemia treatment, children in SR group had
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significantly higher naïve CD4+ and CD8+ T cell counts compared to IR group (p= 0.005 and p=
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0.02). After that, there were no significant differences between the two groups in any of the
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following time-points in any T cell subpopulations studied and there were no differences
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according to the time either.
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In the CD19+ B-cell population, the absolute cell counts of the total CD19+ B-cells, naïve B-
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cells (CD19+CD27low), and the memory B-cells (CD19+CD27+) were notably low during the
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whole study period, and significantly lower in IR group compared to SR group (p < 0.001 in
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each population) (Figure 2). The increase of the B cell populations over the follow-up time was
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statistically significant (p < 0.001) for both of the patient populations.
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Evaluation of humoral immunity
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By the 3-month time-point, all the children in SR group reached the normal age-related levels in
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IgG, IgA, IgM, and IgG subclasses 1-4 (Central Laboratory of Turku and Tampere University
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Hospitals). In the IR group, the normal levels in IgG and IgG subclasses 1-4 were reached by 3-
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month time-point while in IgA and IgM by 6-month time-point. The levels of the
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immunoglobulins and their subclasses significantly increased over the time, except the IgG3
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levels (p  0.05) (Supplemental Table I).
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All the patients had protective levels of antibodies against Clostridium tetani ( 0.1 IU/ml) but
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the antibody levels against Corynebacterium diphteriae ( 0.1 IU/ml) and Bordetella pertussis
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( 50 IU/ml) resulted in non-protective levels in 79% and 76% of the patients vaccinated before
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the leukemia occurred (Department of Microbiology of Turku University by EIA). There were
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no differences between the risk groups (data not shown). Between 6 and 12 months after the
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cessation of leukemia treatment, nine children got pneumococcal vaccination (7 valent
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pneumococcal conjugate vaccine Prevenar), and all of them developed protective levels of the
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antibodies against the vaccine serotypes ( 0.2 g/ml).
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Complement pathways
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Lectine deficiency was found in 4 children out of 27 (15%) by gene test. There was no difference
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in the activity of different complement pathways according to the time or between the two
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groups at any of the time-points. Altogether 8 children had activities under the normal 60% in
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alternative pathway in the first measurement time-point but all these activities normalized during
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the study period (Department of Medical Microbiology and Immunology, University of Turku,
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Finland) [8]. Complement activity was assessed by measuring neoantigen of the terminal
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complex using monoclonal, enzyme-linked antibody (COMPL 300 Wielisa kit, Wieslab, Lund,
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Sweden).
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Statistical methods
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Continuous variables were compared using mixed models repeated measures analysis with time,
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risk group and interaction between time and risk group as predictor variables. Right skewed
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response variables were transformed before the analysis using log-transformation. Comparisons
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between two categorical variables were done using Fisher's exact test. Differences were
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considered statistically significant when p value was < 0.05. Data were analysed using SAS.
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Discussion
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In the present study, we report 28 children with standard or intermediate risk leukemia (ALL)
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completing the conventional chemotherapy and their immunological recovery during the two-
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year follow-up. Substantially, both naïve and memory B cells were severely affected and showed
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slow recovery during the two-year follow-up while T cell compartment showed only minor
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changes. Immunoglobulins and their subclasses, complement activity, and antibodies against
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previously given tetanus and conjugated pneumococcal vaccines were not significantly affected
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or showed rapid recovery after the cessation of leukemia treatment. The rate of the immune
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recovery did not differ markedly between the standard or intermediate risk groups.
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While T-cell recovery in both children and adults after antineoplastic treatment, specifically after
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stem cell transplantations, has been studied intensively, only few reports have been available
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regarding the B-cell compartment. Recently, Wiegering and co-workers found significant
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reduction in B-cells, specifically in naïve B-cells, during and after the standard and medium risk
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ALL treatment 7. The abnormal B-cell distribution and on-going reconstitution was still found
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5 years after the end of the therapy.
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The defects in B-cell immunity have major clinical significance leading to the increased
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susceptibility of viral and bacterial infections, or incomplete antibody response to vaccinations.
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Recently, guidelines on vaccinations in pediatric hematology and oncology patients were
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reviewed by Cesaro and co-workers 9. Their recommendation was to start booster vaccinations
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after 6 months from the end of cancer treatment. Requirement for a successful revaccination is
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sufficient B cell recovery and presenting CD4 T-helper cells 9-11. On the other hand,
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Lehrnbecher et al suggest that children with ALL should be revaccinated with non-live vaccines
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as early as 3 months after chemotherapy 10. In our study, T-cell compartment was relatively
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well preserved but it took 3 to 6 months for the B-cell compartment to recover based on serum
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immunoglobulin levels and normal antibody responses against conjugated pneumococcal
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vaccinations. Because of the limited number of the patients we can not make any
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recommendations for re-vaccination after completing conventional leukemia treatment in
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children.
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Conclusions
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In conclusion, our study showed that the immune recovery after conventional chemotherapy for
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standard and intermediate risk acute lymphoblastic leukemia is marked by B cell depletion but
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otherwise it did not show any severe deficiencies in lymphocyte functions.
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Acknowledgements
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We thank the Foundation of Pediatric Research in Finland, Finnish Cancer Society, and
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Foundation of Cancer Research of Moikoinen, Turku, Finland for their financial support. We
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also thank the research nurse Merja Vuorinen for assisting to carry out the study protocol.
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Declaration of conflicts of interest
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The authors declare no conflicts of interest.
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Figure 1. The absolute cell counts of different CD4+ T cell subpopulations according to the time
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presented separately by standard (SR) and intermediate (IR) risk groups. SR group is shown as
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clear boxes and IR group as dark grey boxes. The following cell populations are shown: naïve
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CD4+ T cells (CD4+CD45RA+CCR7+), central memory CD4+ T cells (CD4+CD45RA-
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CCR7+), effector memory CD4+ T cells (CD4+CD45RA-CCR7-), late effector memory CD4+
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T cells (CD4+CD45RA+CCR7-), Treg (CD4+CD25+ CD127low). The counts are presented as
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109/l in blood for the total CD+ counts. The number of the patients analysed per time-point are
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18/28 (0 m), 23/28 (3 m), 24/28 (6 m), 23/28 (9 m), 22/28 (12 m), 19/28 (18 m) and 14/28 (24
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m). The blue line represents the estimated normal mean value and the red line the lowest range
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of healthy children between 6 and 10 years of age 12 -13.
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Figure 2. The absolute cell counts of total CD19+ B cells, naïve B cells (CD19+CD27-) and
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memory B cells (CD19+CD27) according to the time are presented separately by standard (SR)
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and intermediate (IR) risk groups. The counts are presented as 109/l in blood for the total CD19+
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cell counts. The number of the patients analysed per time-point are 18/28 (0 m), 23/28 (3 m),
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24/28 (6 m), 23/28 (9 m), 22/28 (12 m), 19/28 (18 m) and 14/28 (24 m). The blue line represents
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the estimated normal mean value and the red line the lowest range of healthy children between 6
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and 10 years of age 12 - 13.
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Supplemental Figure 3. The absolute cell counts of different CD8+ T cell subpopulations
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according to the time presented separately by standard (SR) and intermediate (IR) risk groups.
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SR group is shown as clear boxes and IR group as dark grey boxes. The following cell
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populations are shown: naïve CD8+ T cells (CD8+CD45RA+CCR7+), central memory CD8+ T
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cell (CD8+CD45RA-CCR7+), effector memory CD8+ T cells (CD8+CD45RA-CCR7-), late
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effector memory CD8+ T cells (CD8+CD45RA+CCR7-). The counts are presented as 109/l in
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blood for the total CD+ counts. The number of the patients analysed per time-point are 18/28 (0
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m), 23/28 (3 m), 24/28 (6 m), 23/28 (9 m), 22/28 (12 m), 19/28 (18 m) and 14/28 (24 m). The
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blue line represents the estimated normal mean value and the red line the lowest range of healthy
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children between 6 and 10 years of age 12 -13.
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Supplemental Table I. The median peripheral blood cell counts and immunoglobulin levels
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including the subclasses according to the time (0, 3, 6, 9, 12, 18, and 24 months after cessation of
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the leukemia treatment) of standard risk (SR) and intermediate risk (IR) children with leukemia.
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The ranges are presented within ( ). The n – value represents the number of the samples per time-
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point (some patients had more than one sample).
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