ALBERT

Description: Abstract 755 Second hematologic malignancies in non-syndromic children without a pronounced family history for cancer may be mistaken for relapses or therapy-related malignancies. Recently, we characterized diagnosis and presumed relapse samples of 22 patients with very late disease recurrences (〉2.5 years), and identified 8 patients with leukemic presentations that were fully discordant at the level of TCR-rearrangements and DNA copy number aberrations (J Clin Oncol 2011; 29:1643-9). One of these patients showed a germline deletion comprising the recombination activating genes RAG1 and RAG2, and regulatory sequences of LMO2, genes frequently affected somatically in T-ALL, suggesting a genetic predisposition to leukemia. In the current study, we performed exome sequencing to assess whether consecutive leukemic presentations in such patients are indeed fully discordant, also at the sequence level, and to identify candidate pathogenic germline variants that point at a genetic predisposition. We sequenced the exomes in samples obtained from 2 consecutive leukemic presentations, and intermittent complete remissions, from 2 patients with very late disease recurrences (〉2.5 years) and discordant leukemic presentations. We found on average 26,600 variants per exome. Recurrent variants recorded in the dbSNP and/or 1000 Genomes databases, or those present in our in-house database (〉300 exomes) were excluded, resulting in an average of 989 private variants per exome. We divided these variants into 3 groups (i) somatic variants shared between the consecutive leukemic samples but not detected in remission (ii) somatic variants present in only one of the leukemic samples and (iii) germline variants present in the remission samples of the patients. All candidate somatic variants shared between two consecutive leukemic samples were re-sequenced by Sanger sequencing and were shown to be either present in all three samples, and thus originally missed in the remission sample, or falsely detected in one or more leukemic samples. Therefore, we conclude that in both patients no somatic variants were shared between the first and second leukemic presentations, which confirms that these patients suffered from clonally unrelated second T-ALLs. From all somatic variants present in only one of the leukemic samples, we focused on variants in exons or splice junction sites. We found 4 nonsense mutations, 9 frame-shift mutations, 12 in-frame in/dels and 7 non-synonymous missense variants with a high interspecies conservation score (PhyloP〉3.0), mostly affecting genes implicated in oncogenesis like PTEN, TET3, CDKN2C, CD109, and GLRX2. Each leukemic sample harbored 2–11 of these putative deleterious variants. In the germline of the two patients, we identified 314 and 190 non-synonymous unknown variants in exons or splice junction sites, respectively. Among these were 12 nonsense mutations, 7 canonical splice-site mutations, 20 frame-shift mutations, 11 in-frame in/dels and 143 non-synonymous missense variants at highly conserved positions (PhyloP〉3.0). Filtering of these variants for known T-ALL associated genes resulted in several interesting novel candidate predisposing genes such as, among others, RANBP17 and HOXC13. Sequencing of the entire RANBP17 open reading frame in a cohort of 24 sporadic T-ALL samples revealed that this gene was somatically affected in one of them. In conclusion, we confirmed by exome sequencing that consecutive leukemic presentations in patients with late T-ALL recurrences may be fully discordant and thus represent independent leukemia occurrences, most likely caused by predisposing germline abnormalities. Disclosures: No relevant conflicts of interest to declare.

Description: The vast majority of relapses in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients occur relatively early, usually within 2 years from diagnosis. Our previous comparative molecular analyses between diagnosis and relapse in all such “classical” T-ALL showed totally or at least partly identical T-cell receptor (TCR) gene rearrangement patterns at both disease phases. These results confirm that the relapse clone in these patients originated from the original diagnosis clone, which became resistant to the applied treatment. In contrast to these “classical” T-ALL, two patients experienced very late T-ALL recurrences and displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. We hypothesized that such late “relapses” of T-ALL in fact might represent second malignancies in genetically predisposed persons. We investigated 16 T-ALL patients with late relapses, i.e. at least 2.5 years from initial diagnosis. The studies at the DNA level involved detailed comparison of TCR gene rearrangements between diagnosis and relapse (PCR-heteroduplex, sequencing and/or Southern blot analyses), the detection of gene fusions involving the TAL1 gene and/or TCR genes and comparative genomic hybridization (CGH) using high-resolution Agilent arrays. We found evidence of a common clonal origin between diagnosis and relapse in ten of the 16 patients. In one case, no clonal TCR rearrangements were detected neither at diagnosis nor at relapse. In the other five patients TCR gene rearrangement sequences had completely changed between diagnosis and relapse, suggesting a second T-ALL rather than a relapse. Moreover, three of these five patients remained in complete remission after second-line treatment, which is unusual for relapsed T-ALL. In three of the five patients with presumed second T-ALL, CGH arrays showed completely different patterns of genomic aberrations between diagnosis and relapse, while in the remaining two patients the patterns of genomic changes were vastly different, but some aberrations were similar at both disease phases. In eight patients with evidence of a common clonal origin between diagnosis and relapse at least 50% of genomic events revealed by CGH arrays were identical between diagnosis and relapse of T-ALL. We conclude that approximately one-third of late T-ALL “relapses” in fact represent second malignancies. We are currently performing further genomic analyses to identify common genetic events or common genomic features which might be related to predisposition for development of T-ALL.

Description: Abstract 2516 Introduction: In pediatric acute myeloid leukemia (AML) cytogenetic abnormalities are important for prognosis and treatment stratification. Some recurring cytogenetic abnormalities occur so rarely that large collaborative studies are required to define their prognostic impact. t(8;16)(p11;p13) is an aberration reported in

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: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Description: Refractory cytopenia of childhood (RCC) is the most common subtype of childhood myelodysplastic syndrome (MDS). Clinical and laboratory evidence suggest a T-cell mediated pathophysiology in a subset of adult patients with low-grade MDS.Recently, it was shown that in RCC, indicators of an immune-mediated pathophysiology are frequently present. These indicators included the presence of minor paroxysmal nocturnal haemoglobinuria (PNH) clones and T cell receptor (TCR) β-chain variable (Vβ) domain (TCRVβ) skewing. In addition, aberrant immunophenotypes are frequently identified in RCC patients. It is however still unknown to what extend these immune phenomena concomitantly occur in low-grade MDS. Therefore, in the current study we integrated the presence of minor PNH clones, TCRVβ skewing and immunophenotypic aberrancies in a large international cohort of 72 RCC patients. Small PNH clones were present in 31 cases (43%), TCRVβ skewing in 30 cases (42%), and immunophenotypic aberrancies in 43 cases (60%). In only 9 patients (13%) all these three were present. Two parameters were present in 25 patients (35%), and 27 patients (38%) displayed only one parameter. In 11 patients (15%) none of the three characteristics was present. The most prominent parameter was the presence of an aberrant immunophenotype, which was associated with TCRVβ skewing (n=18) or the presence of PNH clones (n= 20) in a subset of the patients. Hypocellular RCC patients with a small PNH clone (〉0.1%) were more likely to respond to IST than PNH negative patients (88% versus 40% of patients responded at six months, respectively, P=0.038). From the current study we could not predict the strongest predictable value of combined parameters, with respect to response on immunosuppressive therapy (IST) in RCC patients, as only 23 patients were treated with IST. We conclude that RCC patients frequently show immunological aberrancies, but that the presence of and combination with PNH and TCRVB skewing is rather heterogeneous. However, our study underscores the fact that in RCC, which might be challenging to diagnose based on morphology only due to hypoplasia, bone marrow immunophenotyping by flow cytometry, combined with either TCRVβ skewing or the presence of PNH clones, may be of important diagnostic value. Future studies in extended RCC series are required to build a prediction model for response to immunosuppressive therapy. Figure 1. Venn diagram depicting the overlap between flow cytometric aberrant immunophenotype, PNH clones and TCRVβ skewing in RCC. Figure 1. Venn diagram depicting the overlap between flow cytometric aberrant immunophenotype, PNH clones and TCRVβ skewing in RCC. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); In Vivo Scribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL.

Description: Background: The anti-acute myeloid leukemia (AML) immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains a humanized anti-CD33 antibody (hP67.6) to facilitate uptake of the toxic calicheamicin-γ1 derivative in CD33-positive AML cells. This putative mechanism implies a critical role for the intracellular accumulation of the toxic moiety for GO-induced cytotoxicity. Indeed, drug efflux by P-glycoprotein (Pgp) mediates resistance to GO and correlates with clinical outcome after GO monotherapy. Furthermore, recent in vitro data obtained in human myeloid cell lines have unequivocally demonstrated a quantitative relationship between CD33 expression and GO-induced cytotoxicity. In light of these findings, we have now re-examined the significance of CD33 expression levels on AML blasts and relationship with Pgp activity for clinical outcome of patients treated with GO monotherapy. Methods: Pre-treatment bone marrow samples from patients enrolled in multicenter phase II protocols evaluating the safety and efficacy of GO monotherapy (generally 2 doses of 9 mg/m2 14 days apart) were used for analysis. Relative CD33 expression was quantified by flowcytometry immunophenotyping using the hP67.6 antibody, and linear fluorescence values used for calculations. Pgp function was cytofluorometrically determined by efflux of the fluorescent dye, DiOC2. Results are presented as mean values and 95% confidence intervals. Unpaired t-tests, Pearson correlations, and logistic regression models were used for statistical analysis. Results: Patients achieving a complete remission (CR) or CR with incomplete recovery of platelet counts (CRp) had statistically significantly higher mean CD33 expression levels (71.20 [57.20–85.19], n=69) compared to non-responders (54.44 [48.38–60.51], n=203; p=0.01). There was an inverse relationship between CD33 expression and Pgp efflux (r=−0.23) and this contributed to responders having a statistically significantly lower mean Pgp efflux (1.40 [1.28–1.52], n=57) compared to non-responders (1.83 [1.72–1.95], n=173; p

Description: Abstract 1482 Inherited loss-of-function mutations in the telomerase complex gene TERT have recently been implicated as risk factors for acute myeloid leukemia (AML) in adults. The telomerase complex is expressed in highly proliferative cells, and is responsible for maintaining telomeres, which cap the ends of chromosomes and protect genomic DNA from eroding during cell division. Impaired telomerase function can result in extremely short telomeres, which limits the proliferative capacity of progenitor cells, and can also lead to chromosomal instability, thus predisposing to malignant transformation. In pediatric AML, the frequency of such mutations, and the association of telomere length with cytogenetic, molecular, and clinical characteristics and outcome, are unknown. In a cohort of 168 pediatric AML patients, we determined the frequency of telomerase complex gene mutations and leukemic cell telomere length, and correlated this with prognostic cytogenetic characteristics (inv(16), t(8;21), MLL rearrangements, normal karyotype, other aberrations), molecular aberrations (CEBPA double mutations, NPM1 mutations, FLT3/ITD, WT1 mutations), clinical characteristics, and outcome. No mutations were present in TERC. Three heterozygous variants in TERT, E327D, T726M, and A1062T, were identified in eight of 168 pediatric AML patients (carrier frequency 0.048). In three of six patients carrying A1062T, remission material was available, in which germ-line origin of the variant was confirmed. The variants E327D and T726M were absent, but A1062T was present in a cohort of 406 geographically matched controls (carrier frequency 0.049). Telomerase activity, as determined by TRAP assay in reconstitution experiments, of the novel E327D variant was unaffected, as was the previously published activity of T726M; the earlier reported activity of A1062T was reduced to 60%. Telomere length of leukemic cells was not associated with age, sex, prognostic cytogenetic subgroup, complex karyotype, or expression levels of telomerase and shelterin complex genes. However, patients carrying the high-risk molecular aberration FLT3/ITD had significantly shorter telomeres than did patients with favorable NPM1 mutation or CEBPA double mutations. Telomere length was not associated with overall survival, event-free survival, or cumulative incidence of relapse. We conclude that, in pediatric AML, telomerase complex mutations do not confer a risk for leukemia development, and although short telomeres correlate with the high-risk molecular aberration FLT3/ITD, telomere length of leukemic cells obtained at diagnosis does not correlate with adverse outcome in this pediatric AML cohort. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Presence of malignant cells in cerebrospinal fluid (CSF) is a risk factor in pediatric acute lymphoblastic leukemia (ALL). Consequently, these patients receive extra intrathecal treatment. We evaluated the concordance between morphological and flow-cytometric (FCM) results, both on freshly analyzed and on stabilized, overnight transported samples. Methods Diagnostic CSF samples of 61 newly diagnosed pediatric ALL patients were divided in two aliquots. One was sent to the local laboratory and processed within a few hours after sampling (lab1). A second aliquot was 1:1 diluted with sterile medium and sent to the reference laboratory (lab2). For all samples, a MGG (May-Grünwald-Giemsa) stained cytocentrifuged slide was morphologically evaluated and two 6-color FCM stainings were performed. Samples were considered positive (CNS2) by MGG if at least one blast was seen and by immunophenotyping if a cluster (≥ 10 events) of ALL cells was detected. Results Comparison of morphological data between both laboratories showed concordance in 33 samples (53%). In 20 of the 28 discordant samples only 1 or 2 blasts were reported by a single laboratory. Comparison of FCM data between laboratories was concordant in 58 samples (95%). Three samples (tumor load 15%-18%) were reported positive by one laboratory only; in two of these

Description: Refractory cytopenia of childhood (RCC) is the most common type of childhood myelodysplastic syndrome (MDS). Because the majority of patients with RCC have a normal karyotype and a hypocellular bone marrow, differentiating RCC from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia ((v)SAA) can be challenging. The histopathological differentiation between RCC and (v)SAA is mainly based on the presence of patchy erythropoiesis with defective maturation and/or the presence of micromegakaryocytes in RCC, and the absence of erythropoiesis and megakaryopoiesis in (v)SAA. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable additional diagnostic tool in differentiating MDS from non-clonal cytopenias in adults, but in childhood MDS, only a limited number of flow cytometric immunophenotyping studies have been reported. Here, we performed the first comprehensive flow cytometric analysis of myeloid and lymphoid progenitor cells, maturing granulocytes, monocytes and erythrocytes in bone marrow aspirates obtained from a large prospective cohort of 81 RCC patients, collected by the European Working Group of MDS in Childhood (EWOG-MDS). RCC was diagnosed according to WHO criteria and confirmed by central review of bone marrow morphology and histology. Bone marrow aspirates obtained from healthy adult individuals (n=9) and pediatric patients with (v)SAA (n=17) or advanced MDS (n=7) were used as controls. We employed a 7-tube, 6-color flow cytometry protocol, and data analysis was performed largely in line with European LeukemiaNet recommendations for flow cytometry in MDS. RCC patients had a strongly reduced myeloid compartment compared to healthy controls, but not as severe as (v)SAA patients. In the majority of RCC patients, immature myeloid and/or lymphoid cells were reduced in numbers, but still detectable, while in the vast majority of (v)SAA patients, myeloid blast cells and CD34+B-cell precursors were absent: both cell types were absent in 27 of 81 RCC patients (33%) and in 15 of 17 (v)SAA patients (88%). Furthermore, the number of flow cytometric abnormalities was significantly higher in RCC patients than in healthy controls and in pediatric patients with (v)SAA, but lower than in advanced MDS. The most frequently occurring flow cytometric abnormalities in RCC were the heterogeneous expression of CD71 and CD36 on immature erythroid cells. Two or more flow cytometric abnormalities were detected in 49 of 81 RCC patients (60%), and in 2 of 17 (v)SAA patients (12%). If a diagnosis of RCC was considered if myeloid blast cells and/or CD34+ B-cell precursors were present, or if two or more immunophenotypic abnormalities were detected, 61 of 81 RCC patients (84%) could be correctly classified, using histopathology as gold standard, whereas the specificity of this combination, using (v)SAA as a control group, was 76% (13 of 17 (v)SAA patients). No significant associations were detected between the presence of flow cytometric abnormalities (defined as two or more abnormalities) in RCC patients and age or sex, the presence of HLA-DR15, bone marrow cellularity, transfusion dependency at diagnosis, the presence of a PNH clone, or skewing of the T-cell receptor Vβ chain. Of interest, although only 5 patients with monosomy 7 were included in the present study, all of them displayed at least 2 flow cytometric abnormalities. RCC with monosomy 7 confers a high risk of progression to AML, but histopathologically, no differences can be detected between RCC cases with or without monosomy 7. In conclusion, our results indicate that, although flow cytometric abnormalities in RCC patients are present at a relatively low frequency, flow cytometric immunophenotyping might be a relevant addition to histopathology and cytogenetic analysis in the diagnostic work-up of RCC. Disclosures No relevant conflicts of interest to declare.