The use of all trans-retinoic acid (ATRA) is the basis of treatment of acute promyelocytic leukemia (APL) and represents the paradigm of differentiation therapy. In general, ATRA is well-tolerated but may be associated with a potentially lethal side-effect, referred to as retinoic acid or differentiation syndrome (DS). The cellular and molecular mechanisms of DS are poorly understood and involve changes in the adhesive qualities and cytokine secretion of leukemic cells during ATRA-induced differentiation. As leukocyte extravasation is a key event in DS pathogenesis, we analyzed the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM-1 and Exon 3 (L125V) of PECAM-1 genes with DS development in APL patients treated with ATRA and anthracyclines. DS was diagnosed in 23/127 (18.1%) APL patients at an average of 11.5 days after the start of ATRA. All patients presented respiratory distress associated with increased ground-glass opacity in chest radiographies. Other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). We detected an association between development of DS and the AA genotype at Codon 469 of ICAM-1 (odds ratio of 3.5; 95% confidence interval: 1.2-10.2). Conversely, no significant association was detected between G241R or L125V polymorphisms at Exon 4 of ICAM-1 and Exon 3 of PECAM-1, respectively. Our results suggest that susceptibility to DS in APL patients may be influenced by genetic variation in adhesion molecule loci.

RESUMO -OBJETIVO. A leucemia promielocítica aguda (LPA) apresenta uma boa resposta ao tratamento com o ácido all trans retinóico (ATRA). Entretanto, alguns pacientes desenvolvem uma complicação grave chamada síndrome do ácido retinóico (SAR).

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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. Bone marrow aspirates from four healthy donors and 94 AML patients (50♀, 44♂) at diagnosis were included in the study. Our cohort included 10 patients with acute promyelocytic leukemia (APL), 8 with core binding factor (CBF) leukemias [6 with t(8;21) and 4 with inv(16)], and 74 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] = 3.8835 [0.6804-11.5598] vs. 1.003 [0.7956-1.265], p=0.0243). Similarly, the expression of NSD3 was higher in AML 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. The association between expression levels of NSD-KMT with age, gender, prognosis (favorable vs. unfavorable), presence or absence of FLT3-ITD and NPM1 mutations, and presence or absence of karyotype abnormalities was evaluated. With the exception of NSD3 and presence/absence of karyotype abnormalities, NSD-KMT gene expression levels were higher in AML subgroups when compared to healthy donors, in all above parameters. NSD2 was more expressed in AML when compared to NSD1/NSD2 groups. NPM1 mutations and FLT3 internal tandem duplications (FLT3-ITD) were detected in 19.1% (18/94) of the patients with AML. NSD1/NSD3 were more expressed in FLT3-ITD mutant vs. FLT3 wild-type group. In addition, NSD2 was frequently more observed in patients aged 65 or older. Next, patients were stratified into two groups according to the median value of each NSD-KMT evaluated. We assessed the correlation between age, white blood cells count, hemoglobin, platelets, and blasts percentage at bone marrow and NSD-KMT expression levels. A positive correlation was observec between NSD1/NSD3 and the percentage of bone marrow blasts at diagnosis (r2 = 0.08080/p-value = 0.0084, and r2 = 0.04937/p-value = 0.0410, respectively). NSD3 was also correlated to the higher number of bone marrow blasts (r2 = 0.1219/p-value = 0.0217), followed by a decrease into the platelet count (r2 = 0.09795/p-value = 0.0436). 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 333.023 days (95% CI:158.541-507.505 days) whereas that of patients in the high NSD2 expression subgroup was 817.629 days (95% CI:238.702-1396.555 days) (p=0,633). No significant difference was observed between the overall survival of patients in the high and low NSD1 expression subgroups. Our data suggest a correlation between the overexpression levels of NSD-KMT with histone modifications, suggesting that this modification and not only DNA methylation can contribute to epigenomic changes associated with AML pathogenesis. Citation Format: Virginia Mara de Deus Wagatsuma, Luisa C. A. Koury, Silvia Helena Sánchez, Lorena Lobo Figueiredo-Pontes, Fernanda Borges da Silva, Fernanda Borges da Silva, Adriana Ines Dore, Ana Silvia Gouvea de Lima, Antonio Roberto Lucena-Araujo, Fabiola Traina, Felipe Saldanha-Araujo, Fabio Pittella, Eduardo Magalhães Rego. Nuclear SET domain (NSD) protein lysine methyltransferases (KMT) are higher expressed in acute myeloid leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A39.

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.

Mutations in the tyrosine kinase receptor FLT3 are the most common molecular abnormality in acute myeloid leukemia (AML) being detected in about 30% of AML cases. According to the protein domain altered FLT3 mutations may be classified as juxtamembrane or activation loop. The former are caused by internal tandem duplications (ITD) in exons 14 and 15 and is detected in 20–27% of AML patients. Mutations in the activation loop are mainly due to point mutations in exon 20 and is present in 5–7% of AML patients. Most of these mutations lead to changes in the aspartate in position 835 (D835), which have been detected in about 7% of AML cases. Both types of mutations cause the constitutive activation of FLT3 and are associated with bad prognosis. AML characteristics in Latin America are different from those in Europe and US. Namely, there is a higher frequency of acute promyelocytic leukemia (APL) and the clinical outcome of adult patients with other subtypes of AML treated with standard protocols is poorer. The worse prognosis seems to be related to the biology of the disease, rather than socio-economic features, based on studies of other hematological malignancies. In order to test if a higher frequency or different FLT3 mutations might explain these observations, we performed a screening for mutations in FLT3 using PCR and single strand conformation polymorphism (SSCP) techniques to evaluate exons 12 to 20, which encode for the intracytoplasmatic domains of the protein. Ninety-nine consecutive patients with AML (90 adults and 9 children) and 55 blood donors (controls) were analyzed. Two synonyms mutations that have not been previously described were detected: one in exon 12 (T526T) and the other in exon 17 (G697G). ITD mutations were detected in 23 (23.2%) patients with AML, therefore within the expected frequency based on the studies in developed countries. On the other hand, D835 mutations were absent, and except for a mutation detected in one patient causing the deletion of the aminoacid in the position 836 no other abnormality was detected in exon 20. No mutations were detected in exons 13, 16, 18 and 19. In adults, mutations were more frequent in acute promyelocitic leukemia and in women. There were no associations between FLT3 mutations and CBC values. Interestingly, FLT3 mutations were less frequently detected in patients whose leukemic cells expressed the CD56 marker. As described for other countries, overall survival was worse in patients with FLT3 mutations. In conclusion, this study demonstrates that the frequency of ITD mutations in FLT3 gene in Brazilian patients with AML was similar to the frequency described in the European and North American populations, whereas activation loop mutations were rarely detected, especially those in D835. Thus, our results suggest that Brazilian patients with AML have a distinct profile of genetic abnormalities. In addition, this is the first study to demonstrate a negative association between FLT3 mutations and the expression of CD56 by leukemic blasts. This is a relevant observation, since CD56 expression per se has been identified as prognostic factor by other investigators. Finally, our study demonstrates that the SSCP method may be useful for screening mutations of FLT3 gene.

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