ALBERT

Description: Abstract 4844 Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (〉2%), del(13q) (〉2.4%), del(11q23.3) (〉2.5%), del(17p13.1) (〉3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Financial support: FAPESP (Proc. 07/52462-7). Disclosures: No relevant conflicts of interest to declare.

Description: The Nuclear SET Domain (NSD) Protein Lysine Methyltransferases (KMT) family is composed of three members: NSD1/KMT3B, NSD2/WHSC1/MMSET and NSD3/WHSC1L1 which regulate gene expression through methylation of lysine 36 of histone H3 (H3K36). NSD2 overexpression was reported in multiple myeloma with t(4;14)/IgH-MMSET. NSDs gene expression profile is unknown in acute leukemias, however NSD1 and NSD3 were described to be fused with the nucleoporin 98 gene (NUP98) in rare AML and myelodysplastic syndrome cases and, both fusion proteins were associated with poor prognosis. The aims of the present study were to characterize the expression of NSD-KMTs in patients with AML and healthy controls, to determine if this expression is associated with specific genetic abnormalities and/or with treatment outcome.A total of four healthy donors and 45 AML patients (27♀, 18♂) at diagnosis were included in the study. Our cohort included 8 patients with acute promyelocytic leukemia (APL), 8 with core binding factor (CBF) leukemias [4 with t(8;21) and 4 with inv(16)], and 29 patients with non-APL non-CBF AML. NSD family gene expression was evaluated by qPCR using the comparative Ct method for analysis. A higher expression of the NSD1 gene was observed in AML cells compared to normal bone marrow (BM) samples {median [range] = 3.202 [0.6804-0.096] vs. 1.003 [:0.7956-1.265], p=0.0243}. Similarly, the expression of NSD3 was higher in AML, but the difference was significant only for the comparison between healthy BM and CBF-AML groups {median [range] = 1.070 [0.6360-1.410] vs. 2.719 [1.238-8.830], p=0.0265}. No significant differences were detected in the analysis of NSD2 expression. Considering the three groups of AML patients, no correlation was found between NSD1, NSD2 or NSD3 expression levels and age, gender, leukocyte counts at diagnosis, karyotype (normal vs. abnormal), frequency of specific genetic abnormalities (t(15;17)/PML-RARA; t(8;21)/RUNX1-RUNX1T1; inv(16)/CBFB-MYH11) or percentage of blasts in bone marrow. NPM1 mutations and FLT3 internal tandem duplications (FLT3-ITD) were detected in 29.6% (13/44) and 21% (9/43) of the patients with AML, respectively. We observed a significant increase in NSD1, NSD2 and NSD3 expression in blasts from patients with FLT3-ITD (p=0.0177), but not in those with NPM1 mutations. These differences remained significant when APL cases were excluded from the analysis. Next, patients were grouped according to NSD1 or NSD2 expression. Patients with NSD1 or NSD2 expression higher or lower than the median value (3.25 and 3.16, respectively), showed no significant differences regarding age distribution, leukocyte counts or percentage of blasts in bone marrow at diagnosis, or presence of genetic abnormalities. Regarding the analysis of treatment outcome, patients with non-APL AML were stratified into high and low NSD1 or NSD2 expression subgroups using the criteria above. The median overall survival of patients in the low NSD2 expression subgroup was of 333,023 days [95% CI:158,541-507,505 days] whereas patients in the high NSD2 expression subgroup was of 817,629 days [95% CI:238,702-1396,555 days] (p=0,633). No significant difference observed between the overall survival of patients in the high and low NSD1 expression subgroups. In order to determine if NSD-KMT levels were associated with changes at histone H3 lysine 4 (H4K4) and H3K36 (known to activate gene transcription), as well at histone H3 lysine 9 (H3K9), H3K27, H3K79 and H4K29, associated to regulatory repression, we ran an experiment using Illumina Infinium Methylation 450k arrays. The comparison between normal and leukemic cells revealed specific histone methylation profiles. There is experimental evidence that histone methylation is a prerequisite for DNA methylation and transcriptional regulation, suggesting interplay between histone and DNA methylation. Our data correlate overexpression levels of NSD-KMT with histone modifications, suggesting that this modification and not only DNA methylation can contribute for epigenomic changes associated to AML pathogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1845 Acute Promyelocytic Leukemia (APL) patients present increased bone marrow microvascular density (MVD) compared to normal bone marrow, which has been associated with the aberrant secretion of the proangiogenic factor VEGF by leukemic cells. The APL associated fusion protein PML-RARα is thought to deregulate the TGF-β pathway, through its dominant negative action on cytoplasmic PML, thus down regulating SMAD2/3 signaling, and VEGF transcription. However, PML-RARα expression was associated with increased TGF-β gene transcription and secretion. We used the low molecular weight quinazolinone alkaloid Halofuginone (HF), which has been shown to be a potent TGF-β inhibitor, to test the association between TGF-β/VEGF/angiogenesis. HF inhibited the VEGF secretion by NB4 (an APL cell lineage) and cell proliferation. To determine the effects of HF in vivo, irradiated NOD/SCID mice were transplanted with leukemic cell from hCG-PML-RARα transgenic mice. Twenty-four hours after transplantation, mice were treated with 150μg/kg of HF by intraperitoneal injections for 21 days. All recipients developed leukemia, however the leukemic infiltration of bone marrow and WBC were significantly lower in animals treated HF (4.2 ± 3.89 vs. 20.6 ± 21.9, p