Background
Sideroblastic anemia represents a heterogeneous group of inherited or acquired diseases with disrupted erythroblast iron utilization, ineffective erythropoiesis, and variable systemic iron ...overload. In a cohort of 421 patients with multisystem mitochondrial diseases, refractory anemia was found in 8 children.
Results
Five children had sideroblastic anemia with increased numbers of ring sideroblasts >15%. Two of the children had a fatal course of MLASA1 syndrome (mitochondrial myopathy, lactic acidosis, and sideroblastic anemia SA) due to a homozygous, 6‐kb deletion in the PUS1 gene, part of the six‐member family of pseudouridine synthases (pseudouridylases). Large homozygous deletions represent a novel cause of presumed PUS1‐loss‐of‐function phenotype. The other three children with SA had Pearson syndrome (PS) due to mtDNA deletions of 4 to 8 kb; two of these children showed early onset of PS and died due to repeated sepsis; the other child had later onset of PS and survived as the hematological parameters normalized and the disease transitioned to Kearns–Sayre syndrome. In addition, anemia without ring sideroblasts was found in three other patients with mitochondrial disorders, including two children with later onset of PS and one child with failure to thrive, microcephaly, developmental delay, hypertrophic cardiomyopathy, and renal tubular acidosis due to the heterozygous mutations c.610A>G (p.Asn204Asp) and c.674C>T (p.Pro225Leu) in the COX10 gene encoding the cytochrome c oxidase assembly factor.
Conclusions
Sideroblastic anemia was found in fewer than 1.2% of patients with multisystem mitochondrial disease, and it was usually associated with an unfavorable prognosis.
Acute lymphoblastic leukemia treatment leads to elimination of blasts and stepwise regeneration of normal hematopoiesis. Several studies identified prognostic relevance of minimal residual disease ...(MRD) in bone marrow (BM) before achieving complete remission (Giuseppe Basso et al., J Clin Oncol, 2009). Crucial question is how to assess BM quality at day 15 (d15) of ALL BFM protocols. In ALL BFM 2009 protocol good quality sample is defined as containing more than 2% erythroid precursors (EP) of nucleated cells. EP were defined as CD19neg(orCD7neg)CD45neg.
Two pt cohorts were included in the study. First cohort (Coh2000) consisted of pts treated by AIEOP BFM ALL 2000, n=196 (177 BCP ALL, 19 T ALL, median follow-up 5.4 yrs, range 0.025-10). AIEOP BFM ALL 2000 study was a PCR MRD based protocol (assessment at day d33 and d78) and flow cytometric MRD (FC MRD) was assessed only on research basis at d15. Second cohort (Coh2009) consisted of pts treated by AIEOP BFM ALL 2009, n=331 (292 BCP ALL, 39 T ALL, median follow up 4.8 yrs; range 0.0027-7.6). In Coh2009, both PCR MRD (d33, d78) and FC MRD d15 were used for risk stratification.
We asked following questions:What is the specificity and viability of EP defined by CD45 negativity? Is a definition based on bright expression CD71 more specific?Is the amount of EP different between B and T ALLs and between risk groups defined by FC at d15? What is the overall frequency of low EP at d15?What is the relationship between amount of EP and FC MRD at d15?Is there any prognostic relevance of low EP?
Results:Population of EP was selected based on negativity of CD45 and a lineage marker (CD19 or CD7) among nucleated cells, which were defined as positive by a SYTO nucleic fluorescent dye. We found a high amount of non-viable cells defined by 4′,6-diamidino-2-phenylindole (DAPI) positivity (6.5-96%, median 55%). When we added bright CD71 into the EP definition (EP CD71++), the percentage of DAPI positivity was significantly lower (0-66%, median 9%) (p<0.0001 in both cohorts).There is no difference in amount of EP at d15 between B and T ALL in either of the cohorts. The treatment reduction in SR pts (FC MRD d15<0.1%) was in Coh2009 used only in BCP ALL and we focused in further analyses on BCP ALLs. Overall, the EP were below 2% in 16% and 18% in Coh2000 and Coh2009, respectively. Within risk groups, EP below 2% at d15 occurred more frequently in Standard Risk (SR; 27%) than in non-SR (non-SR; 12%) in both cohorts (p=0.0002). The frequency of low EP appears higher than the expected frequency of technically poor samples. Moreover, it is unlikely that quality of BM aspiration would depend on the risk group of the pt. This further supports the role of normal BM response to presence of leukemic cells on one hand and to therapy on the other one.In both cohorts we found significant positive correlation between amount of EP and FC MRD at day 15 (Coh2000 p-value= 0.0016 (R 0.23); Coh2009 p value <0.0001 (R 0.33)). The correlation was significant in BCP ALLs only (Coh2000 p value=0.008 (R 0.26); Coh2009 p value<0.0001 (R 0.39)). The same significant correlation is observed in BCP ALLs with more precisely defined population EP CD71++DAPIneg (Coh2000 p value=0.04 (R0.18); Coh2009 p<0.0001 (R 0.35)). Part of the Coh2000 was treated with prednisone and part of the pts with dexamethasone between d8 and d28, whereas the entire Coh2009 received prednisone only. However, the frequency of low EP was not different between dexamethasone and prednisone-treated pts, the correlation between FC MRD and EP was significant only in prednisone treated pts (p=0.0003, R=0.33).We focused on SR BCP ALL pts (FC MRD d15<0.1%). We did not find difference in event free survival (EFS) between pts with amount of EP below and above 2%. This result indicates that the pts with low MRD and low EP are not just pts with hemodiluted BM samples.
Conclusion: Sample quality is essential question in the assessment of MRD in BM. Although low EP may indicate poor BM aspiration quality, it may also result from other biological factors. At d15 BCP ALL, these factors include the interaction of normal BM cells with leukemia, patient's risk group, and type of corticosteroid used. EPs should be detected using an erythroid marker, such as CD71. However, new markers of BM quality, less influenced by leukemia treatment, are needed.
Supported by Ministry of Health of CR, grant nr. 15-28525A, NV18-07-00430 and NV18-03-00343; Czech Science Foundation nr. P302/12/G101.
Brüggemann:PRMA: Consultancy; Incyte: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau.
Next generation sequencing has enabled rapid diagnosis of patients with monogenic diseases and discovery of novel disease-causing variants. However, the functional characterization of their role in ...disease pathogenesis still remains challenging. Reduced folate carrier (RFC) encoded by the SLC19A1 gene is one of the main folate transporters in mammalian cells and its deficiency has been shown to be embryonically lethal in a murine knockout model. In humans, only mutations in the proton-coupled folate transporter (PCFT) and folate receptor alpha (FOLR1) have been shown to cause hereditary folate malabsorption leading to folate deficiency, the latter being brain-specific. RFC is also crucial for the uptake of antifolates, such as methotrexate (MTX).
We present a case of 15-year-old male with severe normocytic anemia (Hb 5 g/dL), ineffective erythropoiesis with megaloblastic erythroid precursors in his bone marrow, elevated bilirubin (34 µmol/l), homocysteine (34.7 µmol/l) and mild esophagitis. He responded well to supplementation with vitamin B12 and folate. His blood count and biochemical signs normalized within one month and he experienced no symptoms until the age of 17, when he presented with a second attack of anemia (Hb 7.8 g/dl) that mirrored the previous one and new onset of mild neurological symptoms. He required erythrocyte transfusions every 6 weeks and did not respond to vitamin B12 therapy. Folic acid was added to his therapy at day 265 of his second episode and his blood count and biochemical results normalized within one month. His disease remains stable on regular supplementation with folic acid. Whole exome sequencing of the patient revealed a homozygous in-frame deletion of 3 nucleotides in SLC19A1 gene, leading to a deletion of phenylalanine residue 212 (F212del) located in the central cytosolic loop, that is crucial for the RFC transport function.
HEK293 cell lines transfected with GFP-tagged mutant SLC19A1 gene showed significantly reduced MTX transport ability compared to wild-type transfected cells, in accordance with previously published results showing that loss of RFC transport is one of the mechanisms of MTX resistance. Confocal microscopy did not show any disruption of membrane trafficking of the protein, suggesting the F212 deletion leads to decreased transport capacity.
K562 cell line was then used to create a permanent model of patient's variant by using CRISPR/Cas9 nuclease targeting the SLC19A1 gene at the respective locus and ssDNA exogenous donor carrying the deletion. Single-cell sorting was then used to generate monoclonal populations homozygous or heterozygous for patient's variant as well as populations with complete SLC19A1 knockout. Proliferation assay showed decreased sensitivity to MTX of cells homozygous for the deletion compared to wild type K562 cells (IC50=0.287 µM vs 0.036 µM). However, the sensitivity to MTX of the cells with SLC19A1 knockout was even lower (IC50=2.380 µM) suggesting partially but not fully inactivating character of patient's variant. Interestingly, the cells heterozygous for the variant showed similar sensitivity to MTX to that of wild type K562 cells (IC50=0.038 µM), in concordance with no observed impact on erythropoiesis of patient's parents, who were confirmed to be heterozygous carriers of the variant by Sanger sequencing.
We describe the first case of inborn RFC deficiency in human. Functional analysis of model cell lines with homozygous F212 deletion in RFC confirmed it impacts the function of RFC and causes the anemia in our patient. The partial reduction of the transporter capacity could explain the late onset of the disease and possibility to reverse the phenotype by regular supplementation of folic acid.
Supported by PRIMUS/17/MED/11 and GACR 17-04941Y
No relevant conflicts of interest to declare.
Introduction
Germline mutations in GATA2 were recently identified as causative for several overlapping syndromes: MonoMAC (monocytopenia, mycobacterial infections), DCML (dendritic cells, monocytes, ...B and NK cells deficiency), Emberger syndrome (lymphedema, sensorineural deafness, multiple warts) and familiar myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Of note, GATA2 mutations were also found in children and young adults with “primary” MDS. Aplastic anemia (AA) constitutes an important differential diagnosis to pediatric MDS, particularly in patients with normal cytogenetics.
Because of heterogeneous phenotype of GATA2 mutated patients, defining a set of typical findings would help in their earlier identification and understanding the natural course of the disease. Therefore we aimed to analyze monocytes and lymphocyte subpopulations with the emphasis on B cell lineage by flow cytometry (FC) and polymerase chain reaction (PCR) in all pediatric patients with GATA2 mutation diagnosed in the Czech Republic.
Patients and methods
Eleven pediatric patients were found to harbor GATA2 mutations in the Czech Republic so far. Three mutations were intronic. There was a clear male predominance (9/11). In 7 patients the disease manifested with MDS in childhood, 2 female patients were followed for immunodeficiency and developed MDS in adulthood. One another patient was diagnosed with interstitial lung disease and chronic EBV infection. His brother, carrying the same mutation, has mild neutropenia. Bone marrow (BM) and peripheral blood (PB) samples were analyzed by FC. The level of intronRSS-Kde recombination excision circles (KREC) and T-cell receptor excision circles (TREC) for assessment of proliferation history of B and T cells was examined by PCR. The control group comprised 26 GATA2 wild-type MDS (“other MDS”) patients and 36 AA patients.
Results
Disturbance of B cell compartment was the most frequently observed anomaly in the patients with GATA2 mutation. We observed a decrease of absolute and relative B cell numbers in PB and BM (n=9/11). In BM there was a decrease of immature CD10pos B cells (n=10) with proportional increase of plasma cells. Peripheral blood B cell immunophenotype was shifted towards memory B cells (n=5/7). Presence of normal B cell precursors CD19pos10pos34pos in BM was observed only in 1 patient in part of follow-up samples. Atypical malignant B lymphoblasts were present in another patient, whose MDS quickly progressed to AML with a clear switch to B lymphoid phenotype. Despite significantly reduced number of B cells the levels of IgG were normal in majority of patients. Only 2 patients had IgG hypogammaglobuliemia, in one patient with chronic active EBV infection IgG hypergammaglobulinemia was present. Slightly decreased IgA level was present in 6 patients. Although B cell numbers in other MDS control patients were significantly lower compared to AA, still the decrease was less prominent in comparison with GATA2.
The decrease of immature and naive B cells in patients with GATA2 mutation was reflected in very low level of KREC in PB and BM. Stored newborn dry blood spots from 4 patients were evaluated for TREC and KREC numbers. Strikingly, only one patient had negative KREC levels (the youngest patient from our cohort with MDS diagnosed at age 4). The remaining 3 patients had normal TREC and KREC levels at birth. Thus, the deterioration of de novo production of B cells occurred supposedly postnatally in most patients. Low KREC levels were also present in some patients with other MDS (n=5).
Relative monocytopenia was found in 2 patients, low NK cells were present in 6 patients. T cells were mostly of naive non-activated phenotype.
Conclusions
Changes in B cell compartment are the most characteristic feature in patients with GATA2 mutation. Decreased number of B cells together with a shift towards mature phenotype and decreased level of KREC reflect history of substantial B cell proliferation in an environment of impaired production. This process appears to happen postnatally and resemble normal ageing process, which is accelerated due to progenitor cell impairment. Immunophenotyping is a useful tool in identifying patients for GATA2 sequencing.
Supported by GAUK 802214, IGA NT/14534-3, NT/13462-4, UNCE 204012, GAČR P301/10/1877
No relevant conflicts of interest to declare.
Abstract 1342
Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. The most common subtype of MDS is refractory cytopenia (RC). Both diseases typically exhibit with ...overlapping features and in both disorders dysregulation of immune system variably contributes to the degree of bone marrow (BM) failure. In the diagnostic algorithm plays role also analysis of consecutive BM samples by morphology.
Patients diagnosed between 2005 – 2011 with at least two BM samples analyzed by flow cytometry (FC) before treatment has started and with centrally evaluated BM biopsy according to EWOG MDS criteria were included into the study. We compared first and the last available sample before treatment (immunosupression or stem cell transplantation). By FC we analyzed following parameters: cell subsets (granulocytes, monocytes, lymphoid cells, erythroid precursors), BM precursors (CD34pos, CD117pos), T cells (CD3pos, CD3pos4pos, CD3pos8pos, CD3posHLA DRpos out of all cells, HLA DRpos out of CD3pos/CD3pos4pos/CD3pos8pos cells); B cells (CD19pos, CD19pos10pos, CD19pos45dim to neg, CD19pos34posout of all cells, CD10pos and CD20pos10neg out of CD19pos). In total 22 patients with AA (12 girls, 10 boys, mean age 11 years; 1.1–18 years) and 20 patients with RC (11 girls, 9 boys, mean age 11 years; 3.7–18) were included into the study. Median of time interval between both samples was 139 (1–1343) days in RC and 15 (1–56) days in AA. WT1 expression on mRNA level was analyzed in the sample before treatment with the highest number of CD34pos precursors to avoid blood contamination. All patients were screened by FISH for changes on chromosome 7 and 8. We asked following questions: Are there differences in the parameters in both bone marrow samples between SAA and RC? Is there any different pattern between d0 and before therapy sample between AA and RC? Are there any differences in WT1 expression between AA and RC group?
RC and AA significantly differ in both time points. AA patients have significantly decreased precursors (CD34, CD117); the difference is more pronounced at the later time point. More lymphocytes (both B and T) and less granulocytes are present at later time point in AA patients (p<0.05, Mann-Whitney test). Activation of CD8 cytotoxic T cells according to HLA DR expression is more distinct in AA patients at later time point. The most significant different parameter between RC and AA is a ratio CD19/CD34 also with the significant trend between two time points (Two way ANOVA, p<0.05). WT1 expression is statistically higher in RC patients; the higher expression is associated with presence of monosomy 7.
By FC statistical differences can be identified in both samples (d0 and before treatment) between RC and AA. More pronounced differences are at later time point, which can be explained by further destruction of precursor and myeloid compartment more pronounced in AA patients compared to RC. WT1 expression is typically high in patients with RC and monosomy 7.
No relevant conflicts of interest to declare.
Abstract 4871
Recent WHO 2008 classification introduced a new category named Mixed Phenotype Acute Leukemia (MPAL) for leukemias in which primary lineage cannot be determined by morphology, ...cytochemistry and/or flow cytometry (FC). Acute bilineal leukemia (ABL) is a subtype of MPAL and is defined by presence of distinct myeloid and lymphoid clonal populations simultaneously at diagnosis. No epidemiological data on ABL have been published so far and there are also few data on the origin of distinct leukemic clones. We examined the incidence and biology of ABL cases among children with primary acute leukemia in the period between 1996 and 2011 in the Czech Republic. Morphology and FC were centrally evaluated in all patients. In total 1065 patients were diagnosed (919 ALL, 146 AML); out of them 3 patients were classified as ABL. Two cases had simultaneous presence of distinct B-cell precursor (BCP) and myeloid clones (BCP-My) at diagnosis, one patient had discrete T ALL and myeloid population (T-My). All ABL patients were screened for immunoglobulin (Ig) and T-cell receptor (TCR) clonality, BCR-ABL, MLL gene rearrangements and FLT3-ITD. All three patients had detectable clonality in lymphoid-specific Ig/TCR rearrangements. In pt1 (BCP-My), FLT3-ITD abnormality and in pt3 (BCP-My), BCR-ABL fusion gene were found. Both patients with BCP-My ABL had Ikaros (IKZF1) gene deletion. In pt2 complex karyotype with MLL gene translocation was identified. Using high speed cell sorting we evaluated the presence or absence of previously mentioned changes in separated subpopulations. In patients with BCP-My ABL, identical clonal Ig rearrangements were found in both lymphoid and myeloid clones. Also FLT3-ITD and BCR-ABL aberrations were present in both clones of respective patients. In pt2 (T-My) TCR gene rearrangement was absent in myeloid population. All three patients achieved complete remission (CR) by lymphoid-directed induction treatment followed by switch to myeloid-oriented blocks according Interfant 99, resp. 2006 protocol in pt 1 and 2 and ALL treatment combined with tyrosine kinase inhibitor (TKI) in pt3. Finally all patients underwent allogeneic hematopoietic stem cell transplantation (SCT) in first CR. Pt1 relapsed after SCT as “typical” cALL. However, the plasticity was maintained and the myeloid clone reappeared at day 28 of relapse therapy. Pt2 relapsed as AML with undetectable TCR gene rearrangements. Pt3 is in complete remission 23 months after SCT with detectable low-level MRD and mixed chimerism with repeated lowering MRD after re-administered TKI dasatinib.
ABL is an extremely rare entity in childhood accounting for less than 0.3 % of all acute leukemia cases. Surprisingly, the same genetic changes can be identified in both clonal populations making the “true” ABL even rarer. FC in combination with morphology is the basic method for identifying ABL and should be followed by detailed genetic analysis. The prognosis of ABL patients in our cohort was poor, despite SCT preceded by the application of treatment modalities targeting both lymphoid and myeloid clones.
No relevant conflicts of interest to declare.
Abstract 876
Immunophenotypic instability during early phase of ALL treatment is a frequent observation during flow cytometric minimal residual disease (FC MRD) monitoring. Antigens typically ...involved include CD10, CD20, CD34 and CD45 and these changes do not usually revoke initial disease classification and do not hamper FC MRD detection. We previously described a subtype of B-cell precursor (BCP) ALL with striking immunophenotypic instability towards monocytoid lineage within the first month of therapy (switching ALL-swALL). Blasts expressing both B and monocytoid markers emerged at early time points on days 8 and 15 of treatment while later only those blasts with pure monocytoid phenotype were present. Incidence of swALL in childhood was unexpectedly high (3-4% of all pediatric BCP ALL) as confirmed in two national reference labs. This phenomenon was associated with aberrant expression of CD2 (LFA-2) on diagnostic blasts (Mejstrikova et al, ASH 2010). The leukemic origin of monocytoid blasts was proven by the detection of clone-specific immunoreceptor gene rearrangements (Ig-TCR). No common genetics aberration was found in a cohort of swALL (n=17), MLL gene was always in germline configuration. We found an increased rate of alteration in IKZF1 gene compared to control BCP ALL cases (p=0.012). An expression analysis of the key hematopoietic regulators showed a difference in CEBPα expression, which is an important transcription factor in transdifferentiation of B cells into macrophages (Xie et al., Cell 2004). Expression of CEBPα is increased in swALL compared to other BCP ALL cases (p=0.01) already at diagnosis prior switching, however, the expression is lower compared to AML (p=0.002). We analyzed CEBPα the methylation status of the promoter region of this gene. Demethylation of CEBPα promoter region analyzed by bisulfite sequencing (295bp-594bp of promoter region) was found in 10/12 swALL cases, while it was seen in only 6/28 control BCP ALLs (Fisher test, p=0.0004). The only cases having demetylation in CEBPα were 5/5 BCR-ABLpos and 1/4 ETV6-RUNX1pos. Whole genome ERRBS method (Enhanced Reduced Representation Bisulfite Sequencing) confirmed this methylation pattern of CEBPα in 7 patients (4 swALL,3 BCP ALL). In order to establish an in vivo model to study the underlying molecular mechanisms, we transplanted ALL cells from 7 swALL patients intrafemorally into NOD-SCIDIL2Rgammanull (NSG) mice. Successfulstable engraftment was achieved only in 2 out of 7 swALL cases (28%) (Fisher test, p=0.049).Interestingly in these two cases, the 200 bp promoter region of CEBPα was methylated to some extent at diagnosis and completely methylated after engraftment into mice, suggesting the possibility of a selective advantage in this context. We treated engrafted animals with prednisolone and in both cases we observed demethylation of CEBPα promoter. Because the rate of engraftment of ALL in NSG is usually very high, these observations may indicate that the biology of this particular subset of patient is distinct.
We described a novel subtype of BCP-ALL with the demethylation of CEBPα promoter region, increased CEBPα expression and immunophenotypic shift towards monocytic lineage during first weeks of the therapy. We identified CEBPα as the potential regulator of this lineage plasticity.
No relevant conflicts of interest to declare.
Abstract 1708
Anecdotic observations of B precursor (BCP) ALL patients with aberrant CD2 expression at diagnosis and a significant shift of phenotype towards monocytes during induction treatment lead ...us to investigate the nature, incidence and characteristics of monocytic transdifferentiation.
Patients with a significant shift of blast phenotype (decrease of CD19 associated with increase of CD14 and CD33, Fig) during the induction phase of treatment were labeled as “swALL”. All Czech patients diagnosed with BCP ALL (n=698) during the course of study served as a control group. A Swiss patient with swALL was confirmed in the Prague laboratory and added to the presented cohort.
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In total, 13 patients with swALL have been identified between 09/96 and 05/10. Monocytic nature was confirmed by cytometry (FC) and/or microscopy in all cases. Comprehensive polychromatic FC investigation not only identified typical CD14pos CD19neg monocytic cells coexisting with unequivocal residual leukemic blasts but also cells in intermediate stages that shared B lymphoid and monocytic characteristics (Fig). Although at diagnosis CD14bright monocytic cells were present in low numbers in bone marrow (BM) (mean±SD; 1.7±2.02%, others: 0.57±0.65%, p=0.006) in all 12 analyzed swALL cases, their frequency increased at day 8 BM (17±21%, others: 1.2±1.2%, p<0.000001). Leukemia-specific Ig-TCR rearrangements were confirmed in sorted monocytic cells in 4/6 cases already at diagnosis, in 4/4 cases at day 8, in 3/6 at day 15 and in 3/4 patients at day 33. Especially at day 33 monocytic cells with proven identical Ig-TCR rearrangements were immunophenotypically indistinguishable from normal monocytes and typically FC and PCR MRD gave significantly discordant results. We asked about common genetic background. SNP arrays and MLPA (multiplex ligation-dependent probe amplification) analyses have not identified a common genetic lesion for swALL except for CDKN2A deletion in 4/10 cases and IKZF1 deletions in 5/10 cases. None of the 13 investigated cases was diagnosed as having MLL translocation, non-diploid DNA index, BCR-ABL, E2A-PBX or TEL-AML. CD2 expression was at least partly expressed in all patients, ranging from 13 to 94%, median 72%.
We next asked whether the observed transdifferentiation could be recapitulated in vitro. The diagnostic BM cells from swALL (n=3) or other BCP ALL (n=9) were cultured for 8 days with or without prednisolone (0-0.5-5-10-100μg/ml). Between days 3 and 8 of culture, the number of CD14posCD19dim cells was significantly greater in swALL cells than in other BCP ALL cells (each day, p <0.05). The transdifferentiation was even more pronounced after prednisolone.
The incidence of swALL ranged from 0.99% (5 of 507) (1996-2007, before we started to use 8-color FC screening panel evaluating simultaneously monocytic and B cell lineage between diagnosis and day 33) to 4.3% (7 of 163) in patients diagnosed between 2007-10 and prospectively screened.
Patients with swALL were treated according to the standard protocols for ALL. Eight of 13 swALL had MRD levels at day 33 >5×10-4, 4 of 13 were prednison poor responders. One patient developed an AML with monocytic phenotype 8 months after the diagnosis.
Examples of phenotype plasticity are known from both experimental studies and malignant diseases. None of the previously defined factors (monosomy 7, MLL rearrangements) was detected in our cohort. Despite the striking association with CD2 we have not identified a causal relationship between CD2 and plasticity yet.
SwALL occurs in 1–4% of B precursor ALL. Although the monocytic cells in these patients have little obvious leukemic features both in microscopy and in standard FC, they are derived from the leukemic clone. Prognosis differs, ranging from standard risk to secondary AML with monocytic phenotype. Optimal treatment appears to be the standard ALL treatment but larger cohorts may indicate special approaches to these patients. SwALL presents with a homogeneous molecular genetics, lack of changes previously shown to be associated with immunophenotype instability, and a strong association with initial CD2 expression. In half of the patients IKZF1 deletions are present.
Supported by: P301/10/1877, NS10480-3, NPV2B06064, MSM0021620813, NS/1000-4, GAUK15710, NS/10472-3, NS/10473-3.
No relevant conflicts of interest to declare.
Abstract 3802
Poster Board III-738
Monosomy 7 or del(7q) are frequent cytogenetic abnormalities in children with myelodysplastic syndrome (MDS) and associates with poor prognosis. MDS globally ...affects all cellular subsets in bone marrow and in peripheral blood. We asked whether flow cytometry (FC) can separate individual subtypes of MDS from each other and from aplastic anemia (SAA) and whether in individual subtypes of childhood MDS can separate patients with and without monosomy 7.
In total we analyzed 94 children with centrally analyzed immunophenotype in the reference lab who were diagnosed and treated for MDS or SAA between 1998 and 2009. In total we analyzed 14 patients with refractory cytopenia, 37 patients with advanced forms of MDS (JMML 10, RAEB 25, CMML 2) and 43 patients with SAA. Monosomy 7/del(7q) was present in 17 patients (RC 6, JMML 3, RAEB 8). Analyzed parameters were as follows: B cells, CD10+CD19+, CD19+45dim/neg, CD19+34+, CD19/CD34 ratio, CD34+, CD117 cells, CD34+38dim/neg, CD3+, CD3+4+, CD3+8+, CD3+HLADR+.
We analyzed all parameters using non parametric tests (Mann-Whitney, Kruskal Wallis) and principal component analysis (PCA).
Principal component analysis of all analyzed patients together clearly separates advanced forms of MDS from RC and SAA, the most contributing factor being the number of CD34 and CD117+ cells. In non parametric statistics following factors significantly differ among MDS subtypes and SAA (Kruskal-Wallis): CD19, CD117, CD34, CD3, CD3+4+, CD8+ and CD3+HLADR+. RC and SAA patients are separated mainly by the number of B cells and the CD34:CD19 ratio. In addition, the following parameters differ between RC and SAA (Mann-Whitney): CD34, CD117 and CD3+HLADR+. Unlike the CD34:CD19 ratio, the number of CD19+34+ precursors does not differ between RC and SAA patients. Patients with monosomy 7 do not differ from the remaining patients when all MDS patients are analyzed together or separately in the respective subgroups (RC, non RC, JMML) by PCA or by non parametric statistics.
PCA separates advanced MDS forms from RC and SAA. Advanced forms of MDS are characterized by increased percentage of CD34+ and CD117+ cells compared to RC and SAA patients. The global reduction of B cell progenitor compartment is pronounced especially in non-JMML cases of MDS, whereas SAA patients typically present with isolated reduction of cells at early stages (CD19+34+) of B cell development. Patients with monosomy 7 cluster within the respective disease category, they do not form own cluster in PCA. Supported by MSMT VZ MSM0021620813, MZO 00064203 VZ FNM, MZO VFN2005, IGA NR/9531-3, NPV 2B06064.
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No relevant conflicts of interest to declare.
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