ALBERT

Description: Abstract 2506 Comprehensive identification of leukemia-associated cell surface molecules is required to improve the specificity and sensitivity of flow cytometric analysis of minimal residual disease. A more comprehensive map of leukemia-associated cell surface features could facilitate the selection and subsequent evaluation of new minimal residual disease (MRD) markers as a resource and provide direct cues for new therapeutic targets. Here, we used the chemoproteomic Cell Surface Capturing technology (CSC) to establish a surfaceome map consisting of 807 cell surface detectable proteins detected in an xenograft model derived from diagnostic samples from 19 pediatric patients with acute lymphoblastic leukemia (ALL). Because CSC is based on direct chemical tagging of protein and glycoprotein residues on the surface of living cells, highly viable samples are required to avoid intracellular contaminants. Direct processing of xenograft samples provided optimal conditions for CSC. We included 8 cases with resistant disease based on persistence of minimal residual disease (MRD) during chemotherapy, which represents a group of patients at need for innovative approaches. Comparative analysis of this proteomic dataset showed that CSC recapitulated and expanded the diagnostic immunophenotype of each patient. To select and test for new proteins with potential value for MRD detection within the large set of identified leukemia proteins, the dataset was filtered against gene expression data from sorted populations of the normal human hematopoietic tree according to their relative RNA expression levels in normal hematopoiesis (DMAP, Novershtern et al, 2011, Cell, 144, 296–309). Based on expected low levels of mRNA expression in normal early and late B-cell precursors in the bone marrow, a subset of markers was identified. These included cell surface features that were previously implicated in leukemogenesis, such as IL7R or FLT3, or shown to serve as diagnostic markers for flow cytometry such as CD58, CD99 and CD300A. For a first clinical validation phase, we selected 38 markers based on their frequency in the CSC dataset to evaluate whether they could contribute to a better distinction of leukemic blasts from their normal counterparts. We tested monoclonal antibodies that were available for 15 candidate markers on diagnostic and remission samples from ALL patients for relevant expression on leukemia cells. Data is currently available for 9 markers that were evaluated prospectively on 51 patients enrolled in current clinical treatment protocols. All markers detected leukemia-associated features in at least a subset of the patients. As anticipated, differences in antigen abundance and antigenic shifts under treatment varied from case to case, underscoring the advantage of increasing the number of available markers for MRD detection. Taken together, our surfaceome data provides an unprecedented view at the cell surface landscape of ALL cells including new prequalified candidate MRD markers, which will accelerate the introduction and subsequent evaluation of multiple parameters for leukemia diagnostics. This also constitutes a valuable resource for functional studies and evaluation of new options for therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

Description: We identified a subset of BCP-ALL with switch towards the monocytic lineage within the first month of treatment (swALL)[Slámová et al Leukemia 2014]. During the switch cells gradually lose CD19 and CD34 expression and acquire CD33 and CD14 positivity. We proved clonal relatedness of switched monocytic blasts with the diagnostic leukemic cells based on identical Ig-TCR rearrangements. SwALL cases are not associated with MLL or BCR/ABL1 aberrancies and lack any known genetic markers of lineage ambiguity (detected by FISH or MLPA). We analyzed transcriptomes of swALL samples at diagnosis (n=4) and at d8 (n=4) where the immunophenotypic switching was already apparent as well as control BCP-ALL (n=4). RNA was isolated form either FACS sorted cells or whole BM when blasts constituted 〉80% of cells. For RNA-Seq we used Illumina HiSeq 2000 paired-end or single end sequencing. Raw sequencing data were analyzed using adapted protocol from Anders at al [Anders et al Nature Protocols 2013] and custom scripts. For methylome analysis we used Enhanced Reduced Representation Bisulfite Sequencing (ERRBS)[Akalin et al PLoS Genetics 2012]. ERRBS quantitatively measures DNA methylation at ~3M CpGs genome-wide. Samples from swALL at diagnosis (n=7) and at d8 (n=4) and control BCP-ALL (n=4) were processed. Analysis was performed according to [Akalin et al Genome Biology 2012] and followed with custom analysis in R statistical language. Comparison (generalized exact binomial test) of transcriptomes of B-lineage blasts from diagnosis between swALLs and control BCP-ALLs revealed a number of differentially expressed genes. Among 300 most significantly differentially expressed were KLF4, CEBPD, CLEC12A and CLEC12B (upregulated in swALL) and ANXA5, VPREB1, CD9 and IGHG3 (downregulated in swALL). Hierarchical clustering separated not only swALL and control BCP-ALL, but also swALL cells before and during the monocytic switch. Changes in gene expression during lineage switch included downregulation of ITGA6, Id2, EBF1, CD19, CD34, FLT3, MYB, CD79a, BCR, PAX5, GATA3 and TCF3 genes and upregulation of S100A10, AIF1, CD14, CD33, LGALS1, RNF130 and MNDA. When comparing all three cell types (swALL B cell and monocytic blasts and control BCP-ALL blasts) we concentrated on 1) immunophenotype switch markers and 2) lineage related transcription factors (TF): 1) Both markers typical for B cell blasts (CD19, CD34) decreased during the switch. However while CD19 was expressed in swALL at diagnosis at same levels as in control BCP-ALL, CD34 was overexpressed in swALL compared to BCP-ALL at diagnosis. Both monocytic markers (CD33, CD14) increased their expression during the switch. CD14 showed no difference between swALL and control BCP-ALL at diagnosis. However CD33 was interestingly upregulated in swALL already at diagnosis and continued to rise during the switch. SwALL had therefore deregulated expression of lineage commitment markers already at diagnosis favoring stemness marker CD34 and myeloid marker CD33. 2) B lineage commitment related TFs (EBF1, TCF3, PAX5) were expressed in B lineage blasts in both swALL and control BCP-ALL. However they were all downregulated during the switch. On the other hand myeloid lineage related transcription factor CEBPA is overexpressed in diagnostic B lineage blasts in swALL compared to control BCP-ALL cases. Similarly CEBPD is overexpressed in swALL and its expression further rises during the switch. Other hematopoietic TFs upregulated in swALL cases include KLF4, NANOG and GATA3. To confirm some of the epigenetic markers of swALL cases (demethylation of CEBPA promoter) and to widen epigenetic screening we used ERRBS. While some of the upregulated genes had expectedly hypomethylated promoters in swALL (CEBPA, GATA3) other genes (TCF3, PAX5) had demethylated promoters in all cases. While the whole DNA methylation picture is still a challenge to draw both omics method could clearly separate swALL cases from control BCP-ALL using principal component analysis. In summary we show that immunophenotypic shift is associated with gene expression changes of surface markers, lineage specific transcription factors and other genes. Some of the genes have altered expression already at diagnosis. Expression of some key lineage genes is differentially regulated by DNA methylation. Supported by: GAUK 914613, GAČR P301/10/1877, UNCE 204012, IGA NT13462-4 Disclosures No relevant conflicts of interest to declare.

Description: Key Points Proteomic analysis of the leukemia cell surface reveals new leukemia-associated features with a potential to improve diagnostics. The ALL surfaceome is a resource for systematic functional exploration.

Description: Introduction: Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL? Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p〈 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p

Description: 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. Conclusions: 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. Disclosures: No relevant conflicts of interest to declare.

Description: CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p

Description: Abstract 1708 Introduction: 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 and Design: 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. Results: 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

Description: 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. Conclusion: 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. Disclosures: No relevant conflicts of interest to declare.