ALBERT

Description: PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in B-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human B-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in B-ALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN–expressing mice efficiently developed B-ALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN–regulated gene candidates that establish the molecular bases of the preleukemic state to drive B-ALL initiation. Hence, our study provides a new in vivo model of human B-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

Description: Key Points An accumulation of alterations in epigenetic modifiers and genes in the JAK/STAT pathway likely drives BI-ALCL oncogenesis. Whole exome sequencing of a large series of BI-ALCL demonstrates recurrent mutations in epigenetic regulators.

Description: PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Description: Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK+ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase–positive (ALK+) lymphomas and human ALK+ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ cells, and demonstrate extracellular HMGB-1–stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK+ cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK+ cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK+ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK+ cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK+ lymphoma epidermotropism.

Description: Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23, MYB was a good candidate gene. Our molecular investigations, based on fluorescence in situ hybridization and rapid amplification of cDNA ends, revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together, these results establish, for the first time, a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia.

Description: Abstract 2879 Introduction: Whole genome and exome sequencing studies recently revealed that CLL genome harbors frequent recurrent mutations as NOTCH1, TP53 and SF3B1 genes. Mutational status of these three genes strongly impacted on treatment-free survival (TFS) and overall survival (OS) in pioneer reports. In this retrospective series, we evaluated the relative role of these mutations along with the karyotype in untreated CLL patients, and after immunochemotherapy given first-line. Patients and methods: 164 patients were included (all naïve from chemotherapy), including 108 who further necessitated therapy according to NCI2008 guidelines. Mutational studies (IgVH sequence, NOTCH1, SF3B1, and TP53 genes) were available in 164/164, FISH (del17p and 11q) in 156/164, and conventional karyotype in 140/164. TFS and OS were calculated from diagnosis and first-line treatment or death respectively, progression-free survival (PFS) was calculated in 108 patients who received rituximab-based immunochemotherapy frontline (94 FCR, 14 R-alkylators), from end of therapy to relapse (according to NCI2008 definition). In 60/94 patients, minimal residual disease (MRD) was assessed three months after completion of FCR by 4-color flow cytometry, according to published guidelines for MRD monitoring. Results: Thirty-seven patients (22.6%) have at least one somatic mutation (TP53, SF3B1 or NOTCH1 genes), 22 NOTCH1 mutations (13.4%), 10 SF3B1 mutations (6.1%) and 8 TP53 mutations (4.5%). SF3B1 mutations are located mainly on the exon 14. TP53 mutations are located mainly on the exon 8 (4/9) and double mutation of TP53 was not detected. Three patients had a mutation of NOTCH1 or SF3B1 combined to a TP53 mutation. Conventional karyotype was obtained in 135/140 CLL (96.4%, 5 culture failures) and 116/135 (85.9%) were associated with chromosomal aberrations. Complex karyotype (CK) and balanced translocations involving IgH locus were shown in 23.9% and 8.1% of patients, respectively. NOTCH1 mutations were found significantly associated with unmutated IgVH and trisomy 12, SF3B1 mutations with male gender and advanced Binet stage, both somatic mutations being mutually exclusive. As expected, TP53 mutations associated with del17p. After a median follow-up of 5 years, 108/164 patients had received frontline immunochemotherapy, for a median treatment-free survival (TFS) of 41.6 months from diagnosis of CLL. Only 8/164 had died, for a median overall survival (OS) not reached, but a 10y probability of survival of 92%. On univariate analysis, shorter TFS was correlated with age

Description: Chronic myeloid leukemia (CML) is the paradigm of malignancy treated by targeted therapies by the use of tyrosine kinase inhibitors (TKI), essentially Imatinib, Dasatinib and Nilotinib. Despite their major efficiency, especially as first line therapies, resistance to these drugs develop partly due to genetic instability inherent to CML. BCR-ABL-kinase mutations remain the first cause of resistance, which appears to be due to clonal selection of cells bearing a given mutation under TKI therapies. Amongst these mutations, the “gatekeeper” T315I mutant is a major concern as it confers resistance to all three TKI clinically used and patients with this mutation have a poor prognosis. The inaccessibility of the TKI to the ABL kinase pocket might not be the only “mechanistic” cause of resistance and it has been suggested that T315I-mutated BCR-ABL (Skaggs BJ et al, 2006) could induce a specific phosphoproteome signature. To evaluate this possibility, we decided to determine if a specific gene expression profiling can be associated with T315I-mutated BCR-ABL, as compared to native BCR-ABL. The human hematopoietic cell line UT7 was transfected with retroviral vectors encoding for native BCR-ABL (UT7.11) or BCR-ABL with the T315I mutation (UT7.T315I). The cell lines were characterized by their cell growth, Western blotting and sequencing. UT7.11 cells were sensitive to Imatinib, Dasatinib and Nilotinib as well as to Ponatinib whereas UT7-T315I cells were resistant to all three TKI except for Ponatinib. Affymetrix microarrays were performed in triplicate on each of three groups (UT7, UT7.11, UT7.T315I). The datas were normalized using the dchip software. Bioinformatics analyzes were performed with R software (packages FactoMineR, limma, PAMR) Mev in TM4 software, enrichment analysis with the GSEA software (Broad institute). The principal component analysis (PCA) showed that the overall RNA expression of UT7.T315I was different from that of UT7.11 (native BCR-ABL) and parental UT7. On factorial map, UT7.11 was found more distant from parental UT7 than UT7.T315I. The contrast analysis of the linear model by the algorithm limma between the 3 groups, showed a strong differential signature of UT7.11 as compared to parental UT7 and UT7.T315I (respectively 4792 and 4813 genes). Only 800 genes were found to be differentially expressed between UT7.T315I and parental UT7. In hierarchical clustering, the total signature obtained in limma confirmed a closed profile between parental UT7 and UT7.T315I. Among the results of the limma model, we identified a 286 specific genes signature for UT7.T315I (both different from parental UT7 and UT7.11 and also not regulated between UT7.11 and UT7). This specific list of UT7.T315I was validated with the T315I group sample segregation by different multivariate methods: PCA, hierarchical clustering and non-negative matrix factorization. Among this T315I-specific gene list limma, 34 ZNF family genes were found (11.88%). Predicting class algorithm based on shunkren centroid (PAMR) separated the three group samples with low classification error and a global list of 368 genes: only 75 genes predicted UT7.T315I group and from this list 13 were in the ZNF gene family (13.33%). By the method of gene set enrichment analysis (GSEA), we explored the top 100 ranked genes as upregulated in UT7.T315I by comparing the two other sample groups. This gene set showed a high representation of ZNF family genes (25%). The design of a gene set with ZNF family genes selected showed a positive enrichment of ZNF (NES = +1.35, p-value

Description: Despite tremendous improvements in the outcome of patients with multiple myeloma (MM) in the past decade, high-risk patients have not benefited from the approval of novel drugs. The most important prognostic factor is the loss of parts of the short arm of chromosome 17, known as deletion 17p (del(17p)). A recent publication (on a small number of patients) suggested that these patients are at very high-risk only if del(17p) is associated with TP53 mutations, the so-called "double-hit" population. To validate this finding, we designed a much larger study on 121 patients presenting del(17p) in 〉55% of their plasma cells, and homogeneously treated by an intensive approach. For these 121 patients, we performed deep next generation sequencing targeted on TP53. The outcome was then compared to a large control population (2505 patients lacking del(17p)). Our results confirmed that the "double hit" situation is the worst (median survival = 36 months), but that del(17p) alone also confers a poor outcome compared with the control cohort (median survival = 52.8 months vs 152.2 months, respectively). In conclusion, our study clearly confirms the extremely poor outcome of patients displaying "double hit", but also that del(17p) alone is still a very high-risk feature, confirming its value as a prognostic indicator for poor outcome.

Description: Hypomethylating agents (HMA) azacitidine (AZA) and decitabine are approved low-intensity frontline therapy for AML pts, but patient selection between intensive versus low-intensity therapy remains mater of debate. To date, description of biomarker of response to HMA has brought few convincing findings. Most studies have focused on somatic mutations in candidate epigenetic genes such as DNMT3A, TET2, and IDH1/2 or tumor suppressor TP53 and led to controversies with none of this gene currently used to guide therapy decision in clinical practice. We performed an hypothesis-free, innovative, exome sequencing study to identify predictive biomarkers of outcome of AML pts treated with AZA. From Jan 2007 to Dec 2016, 279 newly diagnosed non-M3 AML pts were consecutively treated with AZA in the regional cancer network ONCOMIP. The median age was 76 yrs (45-93 yrs). Median follow-up was 5.5 yrs. AML was secondary of MDS, MPN or therapy related in 141 pts (50.5%). Karyotype was adverse in 135 pts (49.1%). Median WBC count was 2.7 G/L. Overall, 54 pts obtained CR/CRi (19,4%) and median OS was 10.6 months. Among the 279 pts, we identified a discovery cohort of 49 pts with extreme phenotype consisting in 26 pts achieving CR/CRi versus 23 pts in failure despite at least 3 cycles of AZA, and a validation cohort of 175 pts with baseline DNA sample and evaluable for response. Adverse karyotypes were more frequent in the validation set (30% vs 53% respectively, p=.004), which was likely due to an enrichment of responders pts in the discovery cohort. For exome sequencing of the discovery cohort, libraries were captured from baseline DNA BM samples with Sureselect all-exome V4 kit (Agilent) followed by paired-end, 150bp reads sequencing on a NextSeq sequencer (Illumina). Sequences were aligned with Nextgene and non-synonymous SNV were filtered according to 3 different patterns defined on gene function (nucleotide analogs metabolism/COSMIC census genes/AML genes), read depth, variant allele frequency and recurrence. An elastic-net regularization combining LASSO and Ridge penalties simultaneously (Zou & Hastie, 2005) was used to identify association between variants and clinical response. Using a resampling approach, bootstrap stability selection (BSS) were computed for each variant and only those with a BSS≥75% were selected. The exon capture baits targeted 98% of genes in the Consensus CDS database. A mean coverage depth of 85× per sample was achieved, with 92.5% of targets covered at ≥ 20× depth. From the 3 gene/SNV filtering patterns, bootstrapping of the data obtained with elastic-net regularization identified 4 candidate SNV in C11orf80, DZIP, ZNF543 and MECOM genes. Inspection of the exome-seq data for MECOM revealed that 11 of the 26 individuals in the responders sample had an heterozygous missense variant in the coding sequence of MECOM at position 169098992 (rs7622799; encoding p.Pro120Ser; MAF = 0.13 in the Exome Aggregation Consortium[ExAC]). We screened the 4 candidates SNV by targeted sequencing in the validation set and identified MECOM rs7622799, in 34/175 pts (19.4%). Response rates did not differ between pts with rs7622799 (8.8% CR/CRi) and without rs7622799 (9.2% CR/CRi, p=1.00). We then assessed whether genotype rs7622799 predicted OS in time dependent analysis using a Piecewise Cox model and demonstrated that pts with rs7622799 had improved OS from 3 months after diagnosis compared with non-rs7622799 (HR 0.56 95%CI[0.34;0.93]; p=0.024). The 3 other SNVs were not associated with patient outcome. We used an extreme phenotype study design, to discover that the missense variant rs7622799 of MECOM is correlated in time dependent analysis with the overall survival of older AML pts treated with AZA. MECOM is a complex locus gene which encodes for several isoforms namely EVI1, a very aggressive oncogene when overexpressed in AML, and MDS1-EVI1 (or PRDM3), which has epigenetic function as histone methyltransferase activity. Both isoforms directly interact with epigenetic modifiers (HDAC, DNMT3A). Further analyses are needed to best understand the functional impact of rs7622799. Although our findings could indicate that rs7622799 is associated with outcome of older AML pts in the setting of AZA, it remains unknown if it could be used to stratify pts between intensive and low-intensity therapy. Targeted sequencing of this SNV in a control cohort treated with intensive chemotherapy is ongoing Disclosures Fornecker: Takeda: Honoraria; Servier: Honoraria.

Description: Validated therapies for older pts with AML could rely on intensive or low-intensity strategies. Patient selection for these options remains controversial. There is currently no validated biomarker which can been used to guide therapeutic decision. TP53 mutations which are known to negatively impact AML pts outcome when treated with ICT, have been recently described as a positive prognosis factor for blast clearance with a 10-days regimen of decitabine (Welch, NEJM 2016). To date, it remains unclear whether AML pts with TP53 mutation represent a clinically homogeneous group. Several classification systems of p53 mutant, derived from in vitro or in vivo data, have been validated in solid tumors and aggressive lymphomas as predictors of p53 mutant functional impact or patient outcome. We retrospectively evaluated the impact of TP53 mutational status on the outcome of a real-world cohort of pts, treated frontline with standard doses of azacitidine (AZA). We further hypothesized that functional characterization of TP53 mutations could define a subgroup of pts with specific outcome with AZA From Jan 2007 to Dec 2016, we identified 279 AML pts enrolled in the regional cancer network ONCOMIP registry, treated frontline with AZA. Median age was 76 yrs (45-93), karyotype was adverse in 135 pts (49.1%), including 54 pts with -17 or del17p (19.4%). AML was secondary to MDS in 71 pts (25.4%), to MPN in 24 (8.6%) and therapy related in 46 pts (16.5%). Pts received a median of 6 cycles (1-67). Overall, 54 pts obtained CR/CRi (19.4%) and median OS was 10.6 months (95%CI ,9.7-12.1). For 224 pts with an available bone marrow baseline DNA sample, TP53 mutations were screened with next-generation sequencing on an Illumina® MiSeq sequencer. Sequencing results were filtered with the IARC TP53 mutations database and a variant allele frequency (VAF) 〉10%, strengthening the specificity of the data of this cohort. Of the 224 analyzed cases, 55 cases (24.6%) contained TP53 mutations. Response rates did not significantly differ between TP53mut (21.8% CR/CRi) and TP53wt (17.8% CR/CRi, p=.50) nor between pts with TP53mut and/or -17/del17p (19.1% CR/CRi) and pts without TP53 abnormality (18.6%CR/CRi, p=.93). Median OS was 7.9 months in pts with TP53mut and 12.6 months in TP53wt (p1 TP53 mutation, we selected the mutation with the predicted highest impact: 15 pts had disruptive mutations (i.e. missense mutation in L2/L3 helix of the DNA binding domain or truncating mutation) versus 40 pts with non-disruptive mutations (Poeta M, NEJM 2007), which was not associated with clinical response (25% in CR/CRi vs 27.9% in failure; p=1.00) nor with 6mOS (46.7% vs 55%, respectively; p=.79)Mutant p53 transactivation activity assessed with a 0-100 evolutionary score (Neskey D, Cancer Research 2015), was not associated with response (median score of 79.3[28-90] in CR/CRi vs 73.3 [49-96] in failure, p=1.00) nor with OS (HR 1.01; 95% CI, 0.99-1.03, p=.51).Relative fitness score (on a log2 scale) which was recently reported as a proxy of p53 mutant in vitro and in vivo cell proliferation advantage (Kotler E, Molecular cell 2018) was not associated with response (median score in CR/CRi of 0.094 [-0.79-0.58] vs 0.094 [-2.52-0.84] in failure, p=.68) nor with OS (HR 0.75; 95% CI, 0.45-1.22, p=.24) Overall, the response rate was not influenced by the TP53mut status, but median OS was negatively impacted by the TP53mut status in the entire cohort and in the sub-group of pts with adverse karyotype. None of the mutant p53 classification systems validated in other neoplasms succeed in identifying a subset of AML pts who specifically benefit from AZA suggesting a rather homogenous functional impact of TP53 mutations in this setting Disclosures Fornecker: Takeda: Honoraria; Servier: Honoraria.