ALBERT

Description: Introduction: Interphase FISH on CD138-selected bone marrow cells enables genetic risk stratification in newly diagnosed multiple myeloma (MM), however as MM remains incurable, most centres still treat newly diagnosed MM uniformly, utilising the most active regimens available. At relapse an increasing choice of regimens, coupled with co-morbidities and treatment-emergent toxicities, means no uniform approach is possible. Instead, therapy is tailored to disease and patient related risk factors. In this setting, FISH testing may be particularly useful if not done at diagnosis and to identify progression events that may alter prognosis. Aim: To evaluate the outcome of FISH analysis in consecutive patients with relapsed MM undertaken at our centre: success rate, frequency of abnormalities, incidence of progression events and correlation of FISH abnormalities with treatment outcomes. Methods: FISH analysis was performed on 192 samples from 154 relapsed patients (2012-13). Plasma cells were selected using magnetic CD138 MicroBeads and interphase FISH carried out using probes as recommended by the EMN (Ross et al, 2012). If patients had no prior results, a full FISH MM panel was performed, using probes for t(4;14), t(14;16), t(11;14), deletion 17p (17p-), Chr 1 abnormalities (1p-/1q+) and deletion 13q (13q-). If patients had been previously tested for an IgH translocation (Tx), a progression event panel was used: 1p-/1q+, 17p- and 13q-. Patients underwent FISH testing prior to starting the next line of therapy. Results: 79% of samples were successfully analysed, with analysis limited in 16% and failed in 5%. Common reasons for failure were poor quality/aged slides, insufficient material and poor hybridisation. 17% of patients had no cytogenetic abnormality. The most common abnormality was 13q- (43.1%), followed by 1q+ (41.4%), t(11;14) (18.3%), t(4;14) (12.4%), 17p- (12.0%) 1p- (8.9%), and t(14;16) (5.6%) Progression events were more common in t(14;16) and t(4;14) groups. All patients with t(14;16) and 82% with t(4;14) had an additional genetic lesion. Only 21% of patients with t(11;14) and 54% with no IgH Tx had an additional event. 80 patients (51.3%) had prior FISH results and 13 (16.3%) had developed a new abnormality on the later test. In 9 cases the progression event was 17p-, in 2 it was 1q+ and 2 cases developed 17p- and 1q+. The patients developing 1q+ were previously standard risk, so repeat testing altered risk group. Acquisition of 17p- indicates especially poor outcome, thus in all 13 cases repeat FISH analysis altered risk. Among patients with progression events none harboured t(11;14), 8 (64%) had no IgH Tx, 3 had t(14;16) and 2 had t(4;14). FISH results were correlated with clinical outcome. Patients were stratified as having high risk genetics [t(4;14), t(14,16), 17p- in ≥50% cells, 1p-/1q+] or standard risk [t(11;14), normal cytogenetics]. 63 (41%) patients were high risk, 83 (54%) standard risk, with no information available for 8 (5%). Both groups had received a median of 2 prior lines of therapy. Response rates (≥PR) to the next line of therapy were similar (60.4% standard risk vs 56.0% high risk). PFS from time of FISH was significantly longer in the standard risk group (9.8 months vs 5.9, p

Description: Rearrangements involving the MLL gene at chromosome 11q23 are associated with leukemia and are present in up to 70% of infant leukemias. Loss of heterozygosity (LOH) has been shown for anonymous polymorphic markers at 11q23 in adult leukemias. To study LOH at theMLL locus, we have identified two new polymorphic microsatellite markers: a GAA repeat (mllGAAn) in intron 6 of theMLL gene and a GA (mllGAn) repeat in the 5′ flanking region of the gene, approximately 2 kb upstream of the translation initiation codon. The heterozygosity index of mllGAAn is 0.54, which renders it useful for analyzing LOH. We screened two groups of leukemia patients to study LOH at the mllGAAn marker. Group A (n = 18) was selected on the basis of presentation before 18 months. Cytogenetic and reverse transcription-polymerase chain reaction analysis showed that 9 of these 18 children had translocations involving MLL. No LOH was observed. Group B (n = 36) were randomly selected children who had presented with leukemia between 1993 and 1994. Cytogenetic analysis of this group showed a variety of different chromosomal abnormalities. LOH was shown in 9 of 20 individuals (45%) who were informative. Microsatellite instability (MSI) was demonstrated in 1 of 18 individuals in group A and 5 of 36 individuals (13.9%) in group B. MSI and LOH were observed simultaneously in three individuals. Loss of an allele was confirmed in one individual by fluorescence in situ hybridization. Individuals with MSI or LOH at mllGAAn were selected for analysis at anonymous polymorphic markers D11S1364 and D11S1356, which flank the MLL gene. No LOH or MSI was observed at these markers in those individuals who were informative. These results show that LOH at the MLL gene locus is a common event during leukemogenesis. Furthermore, the presence of MSI at this locus suggests that the region is a hotspot for genetic instability.

Description: Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

Description: Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.

Description: B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1+ acute lymphoblastic leukemia.

Description: Abstract 11 Children with Down's Syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). We have previously identified somatic mutations in R683 of JAK2 in 20% of DS-ALLs (Bercovich et al, Lancet 2008;372:1484). However the cooperating cytokine receptor and other molecular features have been obscured. Here we report that gene expression analysis reveals that DS-ALL is a highly heterogeneous disease not definable as unique ALL subtype. However, 62% (33 of 53) of the DS-ALL samples analyzed were characterized by aberrant expression of the type I cytokine receptor CRLF2 caused by two types of genomic aberrations. CRLF2 encodes one chain of the heterodimeric receptor of the TSLP cytokine involved in inflammation and lymphoid development and has been recently reported to be involved in translocations to the IgH locus or micro-deletions in 3% of ALLs in children without DS. We have confirmed its cell surface expression on primary DS-ALL cells by Flow cytometry analysis. Consisting with a possible role of CRLF2 as a cytokine receptor, all specimens with the R683 mutated JAK2 had high CRLF2 expression. We further show that CRLF2 and R683 mutated JAK2 cooperate in conferring cytokine independent growth of pro-B cells. Furthermore in 3 of 23 DS-ALL patients with wt. JAK2 we identified a novel activating somatic mutation in CRLF2 replacing the juxta-membrane F232 with Cystein. Mutated CRLF2 but not wild type CRLF2 conferred cytokine independent growth of BaF3 cells associated with phosphorylation of STAT5. Aberrant expression of CRLF2 was also associated with a younger age at diagnosis and worse prognosis. Intriguingly, bioinformatic analysis revealed that gene expression of DS-ALLs is enriched with DNA damage and BCL6 responsive genes suggesting the possibility that DS is associated with B-cell lymphocytic genomic instability leading to the genomic aberrations activating CRLF2. Thus DS confers increased risk for genetically highly diverse ALLs with frequent somatic anomaly in CRLF2, whose higher expression further selects for the activating mutations in itself or in JAK2 (figure). Our data also suggests that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract LBA-5 Intra-clonal, mutational complexity is a hallmark feature of cancer and provides the substrate for sub-clonal selection, progression and therapeutic resistance. There is limited insight however into detailed sub-clonal, genetic architecture in cancers and their propagating ’stem’ cells. Childhood acute lymphoblastic leukaemia (ALL) genotypes are characterized by chimeric fusion genes (or hyperdiploidy) coupled with recurrent, copy number alterations (CNA), primarily in genes regulating cell cycle or differentiation. For ETV6-RUNX1-positive cases of B precursor ALL, the temporal sequence of events is known with the fusion gene usually arising as a pre-natal, initiating event. Recurrent CNA, presumed to be functional or ’driver’ mutations, are secondary to gene fusion and probably post-natal in origin. We have interrogated clonal architecture by multiplexed FISH analysis of single cells using probes labelled with three or four distinct fluorochromes. We pre-selected diagnostic samples from 30 ETV6-RUNX1-positive cases that we found to have deletion of ETV6 and CDKN2A or PAX5 or combinations of these. The application of multiple FISH probes allowed us to score all cells (200 per patient sample) for up to six genetic lesions: ETV6-RUNX1 fusion (and multiple copies of the fusion), deletion of the unrearranged ETV6 allele, extra copies of chromosome 21q (via RUNX1 signals), and one or two copy deletions of PAX5 and CDKN2A. The genetic classification of individual cells using this method allowed a designation of sub-clones and the assembly of putative ancestral, or evolutionary, trees. Although sub-clones were previously known to exist at diagnosis of ALL, the extent of sub-clonal diversity revealed by this study was unanticipated and very marked. Sub-clones, of varying sizes (1–90% of total cells), numbered 3 to 14 per case. This must be an under-estimate of genetic complexity as only selected mutations were included in the screen. The common CNA (ETV6, CDKN2A and PAX5 deletions, chromosome 21q gains) arose independently and recurrently in sub-clones and in no preferential order. Matched relapse and diagnosis pairs were available on 5 patients. In all of these, the clones at relapse could be matched to individual sub-clones at diagnosis, with relapse originating from a major or minor subclone at diagnosis. Importantly, the relapse clone often diversified further, reiterating the evolution pathway of the primary clones. These data indicate that sub-clonal diversification does not arise via linear, clonal succession but rather has a complex, branching architecture reminiscent of Charles Darwin’s 1837 evolutionary speciation model. The ancestral trees so revealed in ALL are single time point snapshots and therefore disguise temporal or sequential dynamics. We show that sub-clonal architecture changes in the months leading up to a diagnosis of ALL and is re-ordered by treatment and subsequent relapse. An important prediction derived from this pattern of sub-clonal architecture is that leukaemia propagating or ’stem’ cells in ALL (and other cancers) should themselves be genetically diverse. Preliminary transplantation experiments in NOD/SCID/IL2Rgamma(null) mice show that this is the case, since multiple, genetically distinct sub-clones regenerated in vivo (Figure1). The complexity of genetic architecture in ALL has substantial implications for the cancer stem cell concept and for the efficacy of generic or targeted treatments. Disclosures: No relevant conflicts of interest to declare.

Description: We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Description: Approximately 25% of pediatric B-cell precursor ALL are characterized by the ETV6-RUNX1 fusion and are associated with a favorable prognosis. Monozygotic twin studies with concordant ALL and ‘backtracking’ studies using archived neonatal blood spots established that ETV6-RUNX1 is an initiating event arising prenatally in a committed B-cell progenitor. However, the fusion gene is not sufficient on its own to cause overt leukemia and a number of subsequent studies have now provided strong evidence that additional mutations are essential for the clinical development of ALL. To obtain a detailed understanding of genetic events that drive this subtype of ALL, we carried out extensive genomic analysis in patients with ETV6-RUNX1 ALL. A total of 57 cases with diagnostic (leukemic) cell DNA paired with matched, remission samples as a source of constitutive DNA were used for exome sequencing (n=56) and low-depth wholegenome sequencing for structural variation (SV) analysis (n=51). Integrative analysis of exome and wholegenome data was performed and data were evaluated for recurrent gene mutations, copy number alterations and genomic rearrangements. Signatures of somatic mutation and structural variation were studied for insights into operative mutational processes and exposures that may drive ETV6-RUNX1pathogenesis. Wholegenome profiling identified 524 somatic SVs (average:11, range 0-49) including 34 tandem duplications, 66 inversions, 106 intrachromosomal translocations and 317 deletions. Resolution of the breakpoint junction to the base-pair level was possible for 354 SVs and in 40% of these breakpoints, we identified a significant enrichment of V(D)J recombination signal sequences or motifs that closely approximate these (E-value=3.8x10-116). Correlation with chromatin marks reveals a ten-fold genomewide enrichment of SVs (pT transitions at CpGs and signature B characterized by C〉G and C〉T at TpCs, contributing 56% and 32% of all substitutions respectively. Signature B is consistent with the reported preference of the APOBEC family of enzymes and is often observed in patients presenting with a hypermutator phenotype and dense clustering of mutations. We report the identification of a novel spectrum of somatic mutations in ETV6-RUNX1 ALL and present the first detailed characterization of the genomic landscape of this ALL subtype. Our analysis shows that the critical secondary events to the ETV6-RUNX1 fusion gene formation are genomic rearrangements mostly driven by aberrant V(D)J recombination events targeting of actively transcribed genes during early B-Cell development. This represents a unique mechanism of genomic instability in cancer, that specifically targets the very genes required for normal B-Cell development and survival, which may in part explain the positive therapeutic response in this subtype of ALL. Disclosures: No relevant conflicts of interest to declare.