Bone Marrow Cells Of Children Research Articles

Cytogenomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals TCR rearrangements as predictive factors for exceptional prognosis

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) represents a rare and clinically and genetically heterogeneous disease that constitutes 10–15% of newly diagnosed pediatric ALL cases. Despite improved outcomes of these children, the survival rate after relapse is extremely poor. Moreover, the survivors must also endure the acute and long-term effects of intensive therapy. Although recent studies have identified a number of recurrent genomic aberrations in pediatric T-ALL, none of the changes is known to have prognostic significance. The aim of our study was to analyze the cytogenomic changes and their various combinations in bone marrow cells of children with T-ALL and to correlate our findings with the clinical features of the subjects and their treatment responses.ResultsWe performed a retrospective and prospective comprehensive cytogenomic analysis of consecutive cohort of 66 children (46 boys and 20 girls) with T-ALL treated according to BFM-based protocols and centrally investigated cytogenetics and immunophenotypes. Using combinations of cytogenomic methods (conventional cytogenetics, FISH, mFISH/mBAND, arrayCGH/SNP and MLPA), we identified chromosomal aberrations in vast majority of patients (91%). The most frequent findings involved the deletion of CDKN2A/CDKN2B genes (71%), T-cell receptor (TCR) loci translocations (27%), and TLX3 gene rearrangements (23%). All chromosomal changes occurred in various combinations and were rarely found as a single abnormality. Children with aberrations of TCR loci had a significantly better event free (p = 0.0034) and overall survival (p = 0.0074), all these patients are living in the first complete remission. None of the abnormalities was an independent predictor of an increased risk of relapse.ConclusionsWe identified a subgroup of patients with TCR aberrations (both TRA/TRD and TRB), who had an excellent prognosis in our cohort with 5-year EFS and OS of 100%, regardless of the presence of other abnormality or the translocation partner. Our data suggest that escalation of treatment intensity, which may be considered in subsets of T-ALL is not needed for nonHR (non-high risk) patients with TCR aberrations.

Expression of chimeric genes in bone marrow cells of children with acute lymphoblastic leukemia upon cytostatic therapy

Expression of chimeric genes in bone marrow cells of children with acute lymphoblastic leukemia upon cytostatic therapy

Evaluation of cadaveric bone marrow cells of children from different regions of Kyrgyzstan

Introduction and Aim: The bone marrow is responsible for the supply of oxygen and restores the cells that form the blood of a tissue. The objective is to study the cadaveric bone marrow cells from dead children of different regions of Kyrgyzstan. Materials and Methods: Bone marrow examination was performed on 38 cadavers (Bishkek–11, Kara-Balta–9, Cholpon-Ata–10, and Naryn–8) of children dead due to causes unrelated to immunodeficiency. The cadaveric bone marrow cells from dead children were collected within a day after death. Results: Studies of cadaveric bone marrow cells from dead children in Kara-Balta showed an increase in basophils, eosinophils, lymphocytes, leukoerythroblastosis, and a decrease in myelocytes. Erythroid growth according to the normoblast hematopoiesis was slightly decreased. Megakaryocytes were single or absent (no function) and contained small number of platelets. Conclusion: Cadaveric bone marrow cells taken from dead children who lived near the uranium tailings dump of Kara-Balta, demonstrated an anomaly with the hematopoietic function of the bone marrow, the state of the stroma, the ratio of hematopoietic and adipose tissue, cellular composition, different pathological processes as indicated by myelocytes in comparison to dead children from other regions (Bishkek, Cholpon-Ata, and Naryn).

PB1651 INFERIOR PROGNOSIS OF TLX3 GENE REARRANGEMENTS IN PEDIATRIC T‐ALL

Background:T‐cell acute lymphoblastic leukemia (T‐ALL) is a clinically and genetically heterogeneous disease that comprises 10‐15% of newly diagnosed pediatric ALL. Despite improved survival rates of children with T‐ALL, relapse occurs in almost 20% of patients and is associated with a dismal subsequent outcome. Therefore, it is of vital importance to identify children already early on during treatment who are at increased risk of such an event. Although several clinical and genetic factors were identified, prognostic implication of majority of them remains unclear. Therefore, the patients risk stratification is currently based only on minimal residual disease (MRD) monitoring.Aims:We performed retrospective and/or prospective comprehensive molecular cytogenomic analysis of bone marrow cells of children with T‐ALL with the aim to determine recurrent chromosomal aberrations and to identify those with significant impact on event free (EFS) and overall survival (OS).Methods:Samples of all patients were analyzed at the time of diagnosis with combination of conventional and molecular cytogenomic methods. For detection of the most frequent known chromosomal changes, i.e. rearrangements of TCR loci and TLX3 gene, deletion of CDKN2A and amplification of ABL1, interphase FISH with locus‐specific probes (Dako, Abbott Molecular) was used. MLPA analysis using SALSA MLPA ALL‐IKZF1 probemix (MRC‐Holland) was performed to detect deletions/amplifications of additional genes. Complex chromosomal rearrangements were proved with multicolor FISH and multicolor banding (24XCyte/XCyte Probe Kit; MetaSystems) or array CGH/SNP (SurePrint G3 Cancer CGH + SNP 4x180K, Agilent). For OS and EFS Kaplan‐Meier analysis with Mantel Cox test was done.Results:During the years 1996‐2017 we examined archived material of 68 children with T‐ALL (21 girls and 47 boys, median age 8.25 years), treated according to ALL Interfant and/or BFM based protocols. Median follow up was 9.5 years. In total, chromosomal aberrations were detected in 91% of patients. The most frequent were deletion of CDKN2A gene in 47/68 patients (27x homozygous, 20x heterozygous), rearrangements of TCR loci in 18/68 children (11x TRA/TRD, 7x TRB) and TLX3 gene rearrangement in 15/68 cases. Complex chromosomal aberrations were proved in 14/68 children. All chromosomal changes occurred in various combinations, however TLX3 aberrations never appeared along with TCR loci rearrangements. 48 patients are living in the first (45x)/second (3x) complete remission, 16 patients relapsed, 19 children died. Best outcome (EFS and OS) was associated with TRA/TRD locus translocations (p < 0.05). Conversely, patients with TLX3 rearrangement had significantly worse OS (p < 0.05).Summary/Conclusion:Considering the poor survival after relapse, it is of utmost importance to identify children at an increased risk of this event. MRD monitoring at different time points is a strong prognostic indicator and is used for risk stratification. In addition, rearrangements of TLX3 gene correlated with poor outcome in contrast to TRA/TRD translocations, which correlated with a more favorable course of the disease.Supported by grant RVO‐VFN64165, GACR P302/12/G157.

Expression significance of Toll-like receptor 4 and myeloid derived suppressor cells in children with acute myeloid leukemia

Objective To investigate the expression of Toll-like receptor 4(TLR4) and myeloid derived suppressor cells(MDSC) in bone marrow cells in children with acute myeloid leukemia (AML), and to detect its relationship with the clinical features, the effect of chemotherapy and prognosis. Methods Twenty-nine cases of children with AML were collected from June 2013 to March 2014 in People's Hospital of Wuhan University, in which 11 cases of low-risk group, 10 cases of middle-risk group, 8 cases of high-risk group; and 17 cases of non blood disease was as the control group.The expressions of TLR4 and MDSC were detected by using reverse transcription-polymerase chain reaction(RT-PCR), Western blot methods, immunohistochemical staining, and flow cytometry, respectively, in the bone marrow cells of 29 children with AML. Results The mRNA and protein expression of TLR4 in the initial treatment group was higher than those in the complete remission group(t=3.092, 3.393, all P<0.05). The mRNA and protein expression of TLR4 in the relapse group was higher than those in the complete remission group(t=4.013, 4.279, all P<0.05). The positive expression rates of MDSC in the above 3 groups were(29.77±1.39)%, (5.19±0.65)%, (38.62±3.54)%, respectively, compared with the control group [(1.32±0.27)%] and there was significant difference(all P<0.05). The positive expression rates of TLR4 and MDSC in the initial treatment group, relapse group and complete remission group were significantly higher than those in the control group, with significant differences (initial treatment group TLR4: t=3.559, P<0.05; MDSC: t=3.727, P<0.05; relapse group TLR4: t=4.043, P<0.05; MDSC: t=4.125, P<0.05; complete remission group TLR4: t=2.798, P<0.05; MDSC: t=3.469, P<0.05). Pearson rank correlation analysis showed that there was a positive correlation between the expression of TLR4 and MDSC (r=0.673, P<0.01). Conclusions The expressions of both TLR4 and MDSC play an important role in onset, progression, curative effect and prognosis in children with AML, and the two may play an importment role in synergistic effect. Key words: Acute myeloid leukemia; Toll-like receptor 4; Myeloid derived suppressor cells

Further Proof for an Unpopular Concept: A Single Cell From Bone Marrow Can Serve as a Stem Cell for Both Hematopoiesis and Osteogenesis

In science, unpopular concepts must be proven time and again. The article by Hofmann et al. in this issue of Molecular Therapy provides further proof for the unpopular idea that a single cell from bone marrow can serve as a stem cell for both hematopoiesis and osteogenesis.1 Moreover, Hofmann et al. establish that this single cell is actually an old friend: the long-term repopulating hematopoietic stem cell (LTR-HSC). These observations also suggest, but do not conclusively prove, the interesting idea that the same LTR-HSC can provide both the HSC and the microenvironmental niche required for the HSC to self-replicate and differentiate. Such a dual function can easily explain why an LTR-HSC is a long-term repopulating cell. The study provides new evidence for a refreshingly simple concept that probably should have occurred to us earlier.

Treatment Intensification Improves Outcome in Children with iAMP21 positive ALL

AbstractAbstract ▪656▪This icon denotes a clinically relevant abstractChromosomal abnormalities present in the bone marrow cells of children with acute lymphoblastic leukaemia (ALL) are used to assist diagnosis and define risk groups that guide therapy. One such primary genetic aberration is intrachromosomal amplification of chromosome 21 (iAMP21) which is defined as the presence of 3 or more extra copies of the RUNX1 gene on a single abnormal chromosome 21. FISH with probes targeting the RUNX1 gene is the commonly used detection method. Approximately 2–3% children with ALL harbour iAMP21 and it is associated with older age (median 10 years) and a low white cell count (WCC, <20×109/L).Among 28 patients treated on the MRC ALL97 trial between 1997 and 2002, 27 (96%) achieved a complete remission (CR), 22 (81%) relapsed and 13 (46%) died. The event free survival (EFS), relapse rate (RR) and overall survival (OS) rates at 5 years were 29% (14–46%), 70% (53–86%) and 67% (47–82%), respectively. There was no evidence that National Cancer Institute (NCI) high risk (HR) and standard risk (SR) patients had differential outcomes and the RR appeared constant with time (figure). On the basis of this increased risk of relapse, we chose to stratify these patients to the most intensive arm of the ALL2003 protocol (regimen C) which included augmented Berlin-Frankfurt-Munster (BFM) consolidation, two blocks of escalating Capizzi maintenance and a double delayed intensification. First remission transplants were recommended for patients who were slow early responders (SER) or minimal residual disease (MRD) positive (>0.01%) at day 29. Due to a relatively high transplant related mortality, transplants were restricted to patients not in CR by day 29 after June 2008.Prospective FISH screening of 2575 patients treated on ALL2003 between January 2003 and July 2011 identified 53 (2%) with iAMP21. The cohort showed a female predominance (30:23), had a median age of 10 years and 47 (89%) patients had a WCC <20×109/L. Follow-up information was available for 52 (98%) patients with a median follow-up time of 4.9 years. The majority of patients (48/52, 92%) were transferred to regimen C as directed by the protocol. At least 2 of the remaining 4 patients did not transfer to regimen C because of induction toxicity problems. Treatment response was measured at day 15 or day 8 according to whether patients received a 3 (NCI SR) or 4 (NCI HR) drug induction, respectively. Whilst none of the SR patients were SER, 13/28 (46%) HR patients had >25% blasts at day 8. MRD was assessed in 45 patients: 23 (51%) were positive (>0.01%) and 22 (49%) were negative (<0.01%) at day 28. All patients achieved a CR, 6 (12%) have subsequently relapsed whilst 4 (8%) died in first remission following a transplant from infection (n=3) or transplant-related complications (n=1). One patient relapsed and died after receiving a transplant; so in total, 5 (10%) patients have died. Among 20 (38%) patients who received a transplant in first CR, 18 (90%) were either MRD positive at day 28 or a SER. The EFS, RR, OS rates at 5 years were 78% (61–88%), 16% (7–34%) and 89% (76–95%), respectively (figure). There were no significant differences in outcome according to sex, age, WCC, NCI risk status or MRD levels at day 28. Interestingly, among the 5 relapses who had MRD measured at day 28 only 1 was positive (>0.01%); although 2 more had borderline levels (>0.005%).A comparison between the outcome of iAMP21 patients treated on ALL97 and ALL2003 showed a statistically significant decrease in RR (p<0.0001) with significant increases in EFS (p<0.0001) and OS (p<0.01). The key endpoint was the proportion of patients suffering a relapse which decreased nearly seven-fold (81% to 12%) between the two trials. Importantly none of the ALL2003 relapses occurred on treatment and all bar one were classified as late events (>6 months after the end of treatment). The findings presented herein clearly illustrate the benefit of this treatment intervention in improving survival in these patients who otherwise had an extremely poor outcome when given standard therapy. In light of these results, iAMP21 patients treated on our new trial, ALL2011, will continue to be classified in the high risk cytogenetic subgroup and will receive regimen C. They are recommended for transplant only if they fail to achieve a CR by day 29. This study illustrates how the discovery and characterisation of a disease-specific genetic aberration can be used to tailor therapy more precisely. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Therapeutic Role of Mobilized Bone Marrow Cells in Children with Nonischemic Dilated Cardiomyopathy

Dilated cardiomyopathy is an important cause of congestive cardiac failure in infants and children. Mobilizing hematopoietic progenitor cells is a promising intervention to this deadly disease. Aim. Evaluate granulocyte colony stimulating factor (GCSF) as therapeutic modality in children with idiopathic dilated cardiomyopathy (IDCM). Subjects and Methods. This case-control prospective study was conducted on 20 children with IDCM following up at Cardiology Clinic Children's Hospital, Ain Shams University (group 1) who were compared to another 10 age-, sex-, duration-of-illness-, and systolic-function-matched children with IDCM as control (group 2). They were subjected to history taking, clinical examination, echocardiography, and peripheral blood CD34+ cell assessment before and one week after GCSF intake for 5 consecutive days (by group 1 but not group 2). Results. A significant improvement in echocardiographic data and CD34+-T-cell increase was found in group 1 one week after GCSF intake and for the next 6 months CD34+ T cells percentage of change showed no significant correlation with the that of the left ventricular dimensions and systolic function. Conclusion. Administration of GCSF to children with IDCM resulted in clinical and echocardiographic improvement not correlated to mobilized CD34+ T cells, implying involvement of additional mechanisms over simple stem cell mobilization.

Fas and Fas Ligand Expression In Children with Acute Lymphoblastic Leukemia, Benign Hematological Diseases and Solid Tumors

AbstractAbstract 4853Fas/FasL is a key pathway of cellular apoptosis. Fas receptor is expressed on membranes of both normal and neoplastic cells while, Fas ligand (FasL) mainly on activated T-lymphocytes. Fas-FasL abnormalities have been detected in malignancies and autoimmune diseases and implicated in resistance to treatment. The aim of the study was the determination of the levels of Fas, FasL and their coexpression in bone marrow cells of children with acute lymphoblastic leukaemia (ALL) at diagnosis, during the course of treatment and the comparison with relevant levels in other hematological and neoplastic diseases. Expression levels of Fas and FasL were determined with flow cytometry in children with ALL at diagnosis (ALLd, n=13), on day 15 of treatment (d15, n=6), on day33 (ALLd33, n=6), during consolidation (ALLhr, n=12) and at the end of therapy (ALLet, n=7) as well as in children with Langerhans Histiocytosis (LCH, n=4), cytopenias (Cytp, n=8) and solid tumors without bone marrow infiltration at diagnosis (STd, n=5) and on therapy (STther, n=6). Results:The lowest levels of Fas expression were detected at diagnosis of ALL and were gradually statistical significantly increased until remission of day 33 (ALLd vs ALLd33: 8.02±1.94 vs 24.04±6.11, p=0.035). At consolidation Fas levels were found to be decreased compared to day33 (16.1±4.18) and were again increased at the end of therapy (ALLd vs ALLet: 8.02±1.94 vs 24.96±7.95, p=0.024). On the contrary, FasL levels were gradually decreased and finally increased to similar to diagnosis levels at the end of treatment (ALLd: 4.59±1.41, ALLet: 5.89±1.99). In solid tumors at diagnosis Fas levels were similar to the ones while on chemotherapy (STd vs STth:16.04±2.2 vs 15.19±6.4). The highest FasL levels were detected in the group of STd with the relevant levels on treatment being lower in comparison (STd vs STther: 10.91±3.32 vs 2.92±0.79, p=0.052). In LCH both Fas and FasL levels were found to be as low as at ALL diagnosis. In cytopenias no significant difference was observed between groups for either Fas (11.05±4.49) or FasL (3.01±0.62). As for Fas+FasL+ coexpression no difference was evident between ALLd, ALLet and STd or STther [ALLd (0.73±0.38), ALLet (0.64±0.17), STd (0.68±0.095), STther (0.66±0.38)]. The lowest coexpreesion levels were observed in the group of cytopenias with statistical significant difference compared to STd (0.68±0.095 vs 0.26±0.08, p=0.031). In conclusion, at diagnosis of ALL Fas levels were expressed in lowest levels that were found to be gradually increased at remission and at the end of treatment. This finding probably correlates with the apoptotic process of the leukemia clone and possibly with response to treatment. Disclosures:No relevant conflicts of interest to declare.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL) which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence-quantitative reverse transcription polymerase-chain reaction(FQ-RT-PCR), and combine minimal residual desease(MRD) detection by flow cytometry(FCM) and to study their relationship with treatment response and prognosis of ALL. Methods The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission(CR), 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9∼101 months, with a median of 64 months. Results Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group( P > 0.05), but significantly higher in the recurrence group than that in newly diagnosed disease group and control group(0.50 ±0.55 vs. 0.09 ±0.26 and 0.12 ±0.23, P < 0.05). 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3∼6 years and over 6 years duration(0.63 ±0.56 vs. 0.11 ±0.12 and 0.04 ±0.06, P < 0.01). For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC > 50×10 9 group and WBC<50×10 9 group( P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>10 3 group than that in MRD<10 3 group(0.39 ±0.47 vs. 0.03 ±0.03, P < 0.05). Conclusion The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrow cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration(within 3 years). Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

The significance and correlation of SODD and bcl-2 protein expression in acute leukemia of children

OBJECTIVE To explore the expression of SODD and bcl-2 proteins in bone marrow cells of children with acute leukemia (AL), and to examine the relationship of their expression with the classification, clinical features, therapeutic effect and prognosis for AL patients.

Regeneration pattern of precursor-B-cells in bone marrow of acute lymphoblastic leukemia patients depends on the type of preceding chemotherapy

Immunofluorescence stainings for the CD10 antigen and terminal deoxynucleotidyl transferase (TdT) can be used for the detection of leukemic blasts in CD10+ precursor-B-acute lymphoblastic leukemia (precursor-B-ALL) patients, but can also provide insight into the regeneration of normal precursor-B-cells in bone marrow (BM). Over a period of 15 years, we studied the regeneration of CD10+, TdT+, and CD10+/TdT+ cells in BM of children with (CD10+) precursor-B-ALL during and after treatment according to three different treatment protocols of the Dutch Childhood Leukemia Study Group (DCLSG) which differed both in medication and time schedule. This study included a total of 634 BM samples from 46 patients who remained in continuous complete remission (CCR) after treatment according to DCLSG protocols VI (1984-1988; n = 8), VII (1988-1991; n = 10) and VIII (1991-1997; n = 28). After the cytomorphologically defined state of complete remission with CD10+ and CD10+/TdT+ frequencies generally below 1% of total BM cells, a 10-fold increase in precursor-B-cells was observed in protocol VII and protocol VIII, but not in protocol VI. At first sight this precursor-B-cell regeneration during treatment resembled the massive regeneration of the precursor-B-cell compartment after maintenance treatment, and appeared to be related to the post-induction or post-central nervous system (CNS) therapy stops in protocols VII and VIII. However, careful evaluation of the distribution between the 'more mature' (CD10+/TdT-) and the 'immature' (CD10+/TdT+) precursor-B-cells revealed major differences between the post-induction/post-re-induction precursor-B-cell regeneration (low 'mature/immature' ratio: generally <1.0), the post-CNS treatment regeneration (moderate 'mature/immature' ratio: 1.2-2.8), and the post-maintenance regeneration (high 'mature/ immature' ratio: 5.7-7.6). We conclude that a therapy stop of approximately 2 weeks is already sufficient to induce significant precursor-B-cell regeneration even from aplastic BM after induction treatment. Moreover, differences in precursor-B-cell regeneration patterns are related to the intensity of the preceding treatment block, with lower 'mature/immature' ratios after the highly intensive treatment blocks. This information is essential for a correct interpretation of flow cytometric immunophenotyping results of BM samples during follow-up of leukemia patients. Particularly in precursor-B-ALL patients, regeneration of normal precursor-B-cells should not be mistaken for a relapse.

Selective pressure as an essential force in molecular evolution of myeloid leukemic clones: a view from the window of Fanconi anemia.

Specific chromosomal deletions are commonly found in bone marrow cells of children with Fanconi anemia (FA) whose disease has evolved to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Identical deletions are found in adults with MDS/AML with a history of exposure to alkylating agents (secondary MDS/AML). While deleted chromosomal regions likely harbor genes encoding proteins with tumor suppressor (TS) function, such genes have not been identified and the environmental forces by which these mutant clones are selected remain unclear. A consistent signaling abnormality in cells bearing mutations of the Fanconi anemia complementation group C (FA-C) gene (FANCC) has revealed a potential selective force. Hematopoietic progenitor cells from patients and mice with FANCC mutations are hypersensitive to the inhibitory effects of IFNgamma and TNFalpha. Consequently, clonal outgrowths in FA likely result from strong selective pressure for stem and/or progenitor cells resistant to these inhibitory cytokines. Additional mutations that inactivate signaling pathways for these inhibitors would create a cell with a profound proliferative advantage over its apoptosis-prone counterparts. Here, we present preliminary evidence supporting a selection-based model of leukemic evolution and argue that MDS in FA patients is a de facto model of secondary MDS in non-FA adults.

PREMATURE CENTROMERE DIVISION OF THE CHROMOSOMES AT THE DIFFERENT STAGES OF ACUTE LYMPHOBLASTIC LEUKEMIA IN INFANTS. 142

The evidence of premature centromere division of the metaphase chromosomes as a new prognostic cytogenetic marker of infant's acute lymphoblastic leukemia (ALL) is suggested. The frequency of PCD cells (premature centromere division of 1-10 chromosomes per cell) and PA (premature centromere division of 50-100% chromosomes) was evaluated in the lymphocytes and bone marrow cells of children with ALL in the acute phase of disease and in remission. PA practically were not found in the lymphocytes of healthy children, but PCD have been registered at a rate no more than 3% of the examined cells. However, in acute phase of ALL the PCD level averaged from 26,9±2,0 per 100 lymphocytes and 40,3±8,6 per 100 bone marrow cells. A significant positive correlation between PCD and blast levels was noted (p=0,85, P<0,01). This allows to assume that PCD cells most probably correspond to blast cells. The PA level averaged from 3,9±1,1 per 100 cells reaching 15,5±8,3 in bone marrow cells. In remission PA practically were not found and the PCD level decreased significantly. The prognosis of disease correlated with PA level in acute phase: If the PA level in acute phase of ALL was 0-8 per 100 cells (average 3,2%), remission was not practically achieved or relapse presented soon. Moreover, if the PA range was 2-70 per 100 cells(average 14,8) a sustained remission was registered. We suggest that PA and PCD levels could be used as prognostic markers of ALL in infants.

Pediatric hematology in Poland: past and present.

Pediatric hematology/oncology gradually developed in Poland in the beginning of the 20th century. The first pediatric hematology books written by M. Erlich were published in 1918 and 1924. In 1939, Jan Raszek-Rosenbusch was the first to use the intramedullary route of injection of living bone marrow cells in children suffering from lymphatic leukemia. The national cooperative chemotherapy group in Poland was formed in 1974. The studies carried out by the group in children with leukemia and lymphoma are documented by publications in international journals.

Assay of lymphokine-activated killer activity generated from bone marrow cells of children with acute lymphoblastic leukemia

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3- hour 51Cr release test. There was a significant cell expansion of 54- fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r- IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.

Assay of Lymphokine-Activated Killer Activity Generated From Bone Marrow Cells of Children With Acute Lymphoblastic Leukemia

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3-hour 51Cr release test. There was a significant cell expansion of 54-fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r-IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.

Low molecular weight B-cell growth factor and recombinant interleukin-2 are together able to generate cytotoxic T lymphocytes with LAK activity from the bone marrow cells of children with acute lymphoblastic leukemia

We are reporting here that low-mol wt B-cell growth factor (LMW-BCGF) and recombinant interleukin-2 (rIL-2) are together able to induce CD3+ cytotoxic T lymphocytes (CTL) with lymphokine-activated killer cell (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). Ficoll-Hypaque (FH)-separated BM cells were obtained from patients with active disease (at diagnosis N = 13, in relapse N = 15) and in complete remission (CR; N = 12). CD3+ cells were removed by Leu-4 antibody and immunobeads. Cells were cultured (10(5) cells/mL) in semisolid media with rIL-2 (100 mu/mL), LMW-BCGF (0.1 mu/mL), and the combination of rIL-2 plus LMW-BCGF, respectively, for seven to ten days. Pooled colonies were harvested for phenotyping. LMW-BCGF plus rIL-2 induced large numbers of CD3+ colonies from CD3- precursors. rIL-2 alone did not induce colony formation. In addition, cells were cultured in liquid media with LMW-BCGF, rIL-2, and the combination of LMW-BCGF plus rIL-2, respectively, for seven to 21 days. They were harvested for phenotyping, and cytotoxicity assays were performed v K562, Raji, and autologous leukemic cells. LMW-BCGF plus rIL-2 induced significant expansion of CD3+ cells from CD3- precursors, and these cells were activated to kill autologous leukemic cells in addition to Raji and K562 cell lines. LMW-BCGF or rIL-2 alone did not induce significant expansion or activation of cytotoxic CD3- cells. Our hypothesis is that LMW-BCGF plus rIL-2 stimulates the proliferation and activation of CD3- precursors from the BM cells of children with acute leukemia to become CD3+ cells that have LAK activity. This finding may have therapeutic implications.

Low Molecular Weight B-Cell Growth Factor and Recombinant Interleukin-2 Are Together Able to Generate Cytotoxic T Lymphocytes With LAK Activity From the Bone Marrow Cells of Children With Acute Lymphoblastic Leukemia

We are reporting here that low-mol wt B-cell growth factor (LMW-BCGF) and recombinant interleukin-2 (rIL-2) are together able to induce CD3+ cytotoxic T lymphocytes (CTL) with lymphokine-activated killer cell (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). Ficoll-Hypaque (FH)-separated BM cells were obtained from patients with active disease (at diagnosis N = 13, in relapse N = 15) and in complete remission (CR; N = 12). CD3+ cells were removed by Leu-4 antibody and immunobeads. Cells were cultured (10(5) cells/mL) in semisolid media with rIL-2 (100 mu/mL), LMW-BCGF (0.1 mu/mL), and the combination of rIL-2 plus LMW-BCGF, respectively, for seven to ten days. Pooled colonies were harvested for phenotyping. LMW-BCGF plus rIL-2 induced large numbers of CD3+ colonies from CD3- precursors. rIL-2 alone did not induce colony formation. In addition, cells were cultured in liquid media with LMW-BCGF, rIL-2, and the combination of LMW-BCGF plus rIL-2, respectively, for seven to 21 days. They were harvested for phenotyping, and cytotoxicity assays were performed v K562, Raji, and autologous leukemic cells. LMW-BCGF plus rIL-2 induced significant expansion of CD3+ cells from CD3- precursors, and these cells were activated to kill autologous leukemic cells in addition to Raji and K562 cell lines. LMW-BCGF or rIL-2 alone did not induce significant expansion or activation of cytotoxic CD3- cells. Our hypothesis is that LMW-BCGF plus rIL-2 stimulates the proliferation and activation of CD3- precursors from the BM cells of children with acute leukemia to become CD3+ cells that have LAK activity. This finding may have therapeutic implications.

Proliferation of normal and leukemic Tdt + bone marrow cells in man

Proliferative characteristics of lymphoid cells with detectable amounts of nuclear terminal deoxynucleotidyl transferase (TdT) in normal or regenerating non-leukemic bone marrow of children were assessed by sequential immunological and cytokinetic studies on single cells and compared to those of TdT + cells in the bone marrow of children with non-T, non-B acute lymphoid leukemia (ALL) at diagnosis or in remission. The median labelling index (LI) of non-leukemic TdT + cells was 21.3% (range 13.2 to 29.7%). The LI of non-leukemic TdT +Ia + cells (range 18.2 to 32.6%) was always higher than that of non-leukemic TdT +Ia + cells (range 0 to 5.3%). Leukemic TdT + bone marrow cells of children with previously untreated non-T, non-B ALL had a LI significantly ( p < 0.001) lower (median 6.1%; range 1.4 to 19.7%) than the LI of non-leukemic TdT + cells. In patients with non-T, non-B ALL in remission neither the percentage of TdT + cells nor the LI of TdT + cells appeared to be useful for detecting early relapse.