INTRODUCTION. Chromosome banding analysis (CBA) is the gold standard to identify complex karyotypes (CK; ≥3 chromosomal aberrations in the same clone). CK are predictors of poor prognosis and ...treatment refractoriness in patients with chronic lymphocytic leukemia (CLL). Patients with CK (15% at diagnosis) constitute a heterogeneous subgroup with highly variable clinical course. Recent studies that aim to refine CK definition in CLL suggest that ≥5 is the number of anomalies detected by CBA that better predicts an impaired outcome (Baliakas et al, 2019). Molecular techniques as genomic microarrays also detect genomic complexity (GC). A recent multicentric ERIC study (Leeksma et al, ASH 2017) identified that patients with ≥5 copy number alterations (CNA) detected by microarrays are associated with an adverse outcome. However, risk stratification regarding genomic complexity assessed by CBA and microarrays has not been compared.
OBJECTIVES. 1. To compare genomic complexity in CLL defined by CBA vs microarrays; 2. To compare risk stratification based on genomic complexity measured by both techniques.
METHODS. The study cohort included 293 CLL patients from 16 European institutions (67% males) with available CBA result at diagnosis or prior to first treatment. The cohort was enriched in patients with CK (n=153, 52%). Tumor DNA extracted from peripheral blood (n=254) or bone marrow samples (n=39) obtained at the time of CBA was hybridized to CGH-arrays (n=12) and SNP-arrays (n=281) platforms. Clinically relevant aberrations 11q-, +12, 13q-, 17p- and CNA ≥5Mb were considered for the anomaly count. Three risk groups were defined using previously suggested cut-off points for CBA and microarrays non-CK/low-GC: 0-2; low/intermediate-CK/GC: 3-4; high-CK/GC: ≥5 (Baliakas et al, Leeksma et al). Groups obtained by both methods were compared and correlated with other clinical and biological data. Time to first treatment (TTT) of patients categorized according to the number of alterations detected by CBA and microarrays was analyzed.
RESULTS. Median number of abnormalities detected was 3 (range: 0-19) by CBA and 2 (range: 0-18) by microarrays. When stratified according to previously defined criteria, a moderate agreement was observed between both techniques (κ=0.483, p<0.001). Remarkably, 8/74 (11%) of patients with high-CK were considered low-GC by microarrays while none of the 140 patients with non-CK was classified as high-GC by microarrays (Table 1). Discordances in those 8 cases underestimated by microarrays were due to the presence of chromosome markers or complex rearrangements in the karyotype which were globally balanced or to subclonal aberrations expanded during CBA culture but represented in a minor proportion of the whole sample.
Regarding the prognostic value of genomic complexity and considering the number of abnormalities detected as a continuous variable, CBA and microarrays showed a similar concordance index (C-index) for TTT (0.615 vs 0.609, respectively). When considering all the abnormalities independently of their size or when lowering the cutoff to 1Mb for those non-CLL abnormalities, similar impact on TTT was observed (C-index=0.593 vs 0.616). The three risk groups defined by each method showed significant differences on TTT (Figure 1, p<0.001). In discordant cases, significant differences on TTT were only observed in cases with high-CK, where low-GC and high-GC showed poor outcome when compared to intermediate-GC group (Figure 2, p=0.009). As genomic complexity category increased in both techniques, a significant increment of del/mutTP53 (CBA: 13% vs 29% vs 62%, p<0.001; microarrays: 16% vs 26% vs 68%, p<0.001) and unmutated IGHV (U-IGHV) (CBA: 49% vs 59% vs 71%, p=0.015; microarrays: 47% vs 68% vs 73%, p=0.001) cases was observed. Of note, among the 8 high risk patients underscored by microarrays, 3 showed del/mutTP53 and 6 showed U-IGHV. Additional techniques, as chromosome painting, are ongoing to confirm microarray results and find an explanation for discordances.
CONCLUSIONS. 1. CBA and microarrays are helpful techniques for assessing genomic complexity in CLL patients; 2. Risk categories established by both methods have a significant impact on TTT although they show a moderate agreement; 3. Discordant cases are being investigated to refine genomic complexity criteria equivalent by both techniques.
ACKNOWLEDGEMENTS. 17SGR437, GLD17/00282, FPU17/00361
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Rigolin:AbbVie: Speakers Bureau; Gilead: Speakers Bureau; Gilead: Research Funding. Gimeno:JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Bosch:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Cuneo:Amgen: Honoraria; Abbvie: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Abstract
The chromosomal translocation t(7;11)(p15,p15), that results in the oncogenic fusion protein Nup98-Hoxa9 (NH), appears in 1% of patients with AML and is associated with very poor prognosis ...and short overall survival. Despite the large severity of the leukemia induced by this fusion protein, the oncogenic events triggered by NH are poorly understood, although a potential role as an aberrant transcription factor has been proposed. We have generated a human Hematopoietic Progenitors (hHP) cellular model expressing NH constitutively to identify the molecular mechanisms supporting the malignancy of this fusion protein, facilitating the search for therapeutic targets.
We identified the DNA binding sites of NH by performing ChIP-seq experiments, which were validated by qRT-PCR analysis on ChIP selected DNA and Luciferase assays. Expression profiling was performed in hHP-NH and co-Immunoprecipitations (Co-IPs) were done to demonstrate the interaction of NH with different transcriptional regulators. Specific drug sensitivity of the hHP-NH model was assessed in cell proliferation assays.
Our work provides the first description of the DNA binding sites of NH, most of which are regulatory regions of genes involved in the development of AML. In particular, we demonstrate that NH induces the overexpression of MEIS1, HOXA9 and PBX3, transcription factors forming an activator complex that is a key element in the leukemic onset driven by other chromosome rearrangements. Interestingly, we show that NH directly interacts with this complex through Pbx3. To evaluate the biological relevance of the interaction of the MEIS1-HOXA9-PBX3 complex with NH, we have analyzed the sensitivity of hHP-NH to the HXR9 peptide (an inhibitor of the HOXA9-PBX3 interaction). Supporting our hypothesis, we observed an inhibitory effect on hHP-NH viability after HXR9 treatment. Finally, by combining the expression profile data from hHP-NH and the ChIP-seq results using GSEA analysis, we show that NH is able to induce both overexpression and down-regulation of its target genes. To provide evidences of the activator-repressor role of NH, we performed different Co-IPs that demonstrated its direct interaction with both p300 (transcriptional activator) and HDAC1 (transcriptional inhibitor).
Taken together, we show that the direct overexpression of the complex MEIS1-HOXA9-PBX3 is one of the pathogenic mechanisms induced by NH. As expected, the disruption of this complex with the HXR9 peptide in the hHP-NH model has a direct effect on cell viability. Furthermore, we show that NH interacts with this complex via PBX3 and also with p300 and HDAC1. The features and architecture of these interactions need to be further explored, but these findings allow us to consider the use of the HXR9 peptide or some HDAC inhibitors as possible treatments for these patients.
Citation Format: Ana Rio-Machin, Alba Maiques-Diaz, Sandra Rodriguez-Perales, Sara Alvarez, Rocio N. Salgado, Álvaro Eguileor, Raul Torres, Juan C. Ramirez, Juan C. Cigudosa. Interactions of the fusion protein Nup98-Hoxa9 with Pbx3, p300 and HDAC1: widening the targeted therapy window in acute myeloid leukemia (AML). abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2014-472
The aim of the present study was to describe the epidemiological and clinical characteristics of inflammatory bowel disease (IBD), including medical and surgical treatments, in several countries in ...Latin America and the Caribbean.IBD is recognized as a global health problem because its incidence and prevalence have increased significantly over the last few years.This multicenter retrospective cohort study included 4714 patients with IBD diagnosed from 9 countries in Latin America and the Caribbean: Colombia, Cuba, Dominican Republic, Ecuador, Mexico, Peru, Puerto Rico, Uruguay, and Venezuela.Crohn disease (CD) was more frequent in Puerto Rico (71.9%), the Dominican Republic (61.0%), and Peru (53.1%). Ulcerative colitis was more frequent in Colombia (78.6%), Venezuela (78.2%), Mexico (75.5%), Cuba (69.9%), Ecuador (64.1%), and Uruguay (60.9%). The following clinical characteristics were more frequent in the Caribbean: penetrating behavior in CD, steroid dependence, steroid resistance, intolerance to thiopurines, extraintestinal manifestations, surgeries, hospitalizations due to IBD, and family history of IBD. The factors associated with the use of biological therapy were pancolitis in ulcerative colitis, penetrating behavior in CD, steroid resistance and dependence, presence of extraintestinal manifestations, and the need for surgery.This study from Latin America and the Caribbean demonstrated the different epidemiological and clinical characteristics of IBD.
LE Fuentes-Ramirez, R Bustillos-Cristales, A Tapia-Hernandez, T Jimenez-Salgado, ET Wang, E Martinez-Romero and J Caballero-Mellado
Programa de Ecologia Molecular y Microbiana, Centro de ...Investigacion sobre Fijacion de Nitrogeno, Universidad Nacional Autonoma de Mexico, Apdo Postal 565-A, Cuernavaca, Mor., Mexico
Diazotrophic bacteria were isolated, in two different years, from the
rhizosphere and rhizoplane of coffee (Coffea arabica L.) plants cultivated
in Mexico; they were designated as type DOR and type SAd isolates. They
showed characteristics of the family Acetobacteraceae, having some features
in common with Gluconacetobacter (formerly Acetobacter) diazotrophicus, the
only known N(2)-fixing species of the acetic acid bacteria, but they
differed from this species with regard to several characteristics. Type DOR
isolates can be differentiated phenotypically from type SAd isolates. Type
DOR isolates and type SAd isolates can both be differentiated from
Gluconacetobacter diazotrophicus by their growth features on culture media,
their use of amino acids as nitrogen sources and their carbon-source usage.
These results, together with the electrophoretic mobility patterns of
metabolic enzymes and amplified rDNA restriction analysis, suggested that
the type DOR and type SAd isolates represent two novel N(2)-fixing species.
Comparative analysis of the 16S rRNA sequences revealed that strains
CFN-Cf55(T) (type DOR isolate) and CFN-Ca54(T) (type SAd isolate) were
closer to Gluconacetobacter diazotrophicus (both strains had sequence
similarities of 98.3%) than to Gluconacetobacter liquefaciens,
Gluconacetobacter sacchari (similarities < 98%) or any other
acetobacteria. Strain CFN-Cf55(T) exhibited low levels of DNA--DNA
reassociation with type SAd isolates (mean 42%) and strain CFN-Ca54(T)
exhibited mean DNA--DNA reassociation of 39.5% with type DOR isolates.
Strains CFN-Cf55(T) and CFN-Ca54(T) exhibited very low DNA reassociation
levels, 7--21%, with other closely related acetobacterial species. On the
basis of these results, two novel N(2)-fixing species are proposed for the
family Acetobacteraceae, Gluconacetobacter johannae sp. nov. (for the type
DOR isolates), with strain CFN-Cf55(T) (=ATCC 700987(T)=DSM 13595(T)) as
the type strain, and Gluconacetobacter azotocaptans sp. nov. (for the type
SAd isolates), with strain CFN-Ca54(T) (=ATCC 700988(T) =DSM 13594(T)) as
the type strain.
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