High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21

The AML1 gene, situated in 21q22, is often rearranged in acute leukemias through t(8;21) translocation, t(12;21) translocation, or less often t(3;21) translocation. Recently, point mutations in the Runt domain of the AML1 gene have also been reported in leukemia patients. Observations for mutations of the Runt domain of the AML1 gene in bone marrow cells were made in 300 patients, including 131 with acute myeloid leukemia (AML), 94 with myelodysplastic syndrome (MDS), 28 with blast crisis chronic myeloid leukemia (CML), 3 with atypical CML, 41 with acute lymphoblastic leukemia (ALL), and 3 with essential thrombocythemia (ET). Forty-one of the patients had chromosome 21 abnormalities, including t(8;21) in 6 of the patients with AML, t(12;21) in 8 patients with ALL, acquired trisomy 21 in 17 patients, tetrasomy 21 in 7 patients, and constitutional trisomy 21 (Down syndrome) in 3 patients. A point mutation was found in 14 cases (4.7%), including 9 (22%) of the 41 patients with AML of the Mo type (MoAML) (none of them had detectable chromosome 21 rearrangement) and 5 (38%) of the 13 myeloid malignancies with acquired trisomy 21 (1 M1AML, 2 M2AML, 1 ET, and 1 atypical CML). In at least 8 of 9 mutated cases of MoAML, both AML alleles were mutated: 3 patients had different stop codon mutations of the 2 AML1 alleles, and 5 patients had the same missense or stop codon mutation in both AML1 alleles, which resulted in at least 3 of the patients having duplication of the mutated allele and deletion of the normal residual allele, as shown by FISH analysis and by comparing microsatellite analyses of several chromosome 21 markers on diagnosis and remission samples. In the remaining mutated cases, with acquired trisomy 21, a missense mutation of AML1, which involved 2 of the 3 copies of the AML1 gene, was found. Four of the 7 mutated cases could be reanalyzed in complete remission, and no AML1 mutation was found, showing that mutations were acquired in the leukemic clone. In conclusion, these findings confirm the possibility of mutations of the Runt domain of the AML1 gene in leukemias, mainly in MoAML and in myeloid malignancies with acquired trisomy 21. AML1 mutations, in MoAML, involved both alleles and probably lead to nonfunctional AML1 protein. As AML1 protein regulates the expression of the myeloperoxidase gene, the relationship between AML1 mutations and Mo phenotype in AML will have to be further explored. (Blood. 2000;96:2862-2869)     

MICM分型诊断在鉴别M2和M3型急性髓性白血病中的意义

目的：探讨形态学、免疫学、细胞遗传学和分子生物学(MICM)分型诊断在鉴别M2、M3型急性髓性白血病(AML)中的意义。方法：对10例按FAB方案难以区分M2、M3型的AML及2例在基层医院诊断为M3b，用全反式维甲酸(ATRA)治疗未获缓解的患者应用常规细胞遗传学(CC)进行核型分析；以筑巢式逆转录聚合酶链反应(nested—RT—PCR)技术检测PML／RARa及AML1／ETO融合基因转录本；以流式细胞术检测白血病细胞免疫表型。对2例在基层医院诊断为M3b而用ATRA治疗效果不好的病例用间期双色FISH技术检测AML1／ETO融和基因。结果：12例患者中，4例有t(8；21)．AML1／ETO融合基因转录本阳性，确诊为M2；2例有t(15；17)，PML／RARa融合基因转录本阳性，确诊为M3；其他6例患者为正常核型，其中，3例AML1／ETO阳性，确诊为M2；1例PML／RARa阳性，确诊为M3；2例PML／RARa及AML1／ETO均为阴性．1例免疫表型为CD13、CD33^ 、CD34^ 、CD19^ ，最后诊断为M2，另1例免疫表型为CD13^ 、CD33^ 、CD34^ 、CD19^ ．最后诊断为M3。2例行FISH检测的患者AML1／ETO融和基因均为阳性。结论：对形态学无法鉴别M2、M3的AML进一步进行细胞遗传学、分子生物学及免疫学检测，可提高确诊率，并为治疗提供可靠的依据。     

Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia

Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8(+) T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1(+)Tim-3(+) CD8(+) T cells were deficient in their ability to produce IFN-γ, TNF-α, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3(+)PD-1- KO CD8(+) T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8(+) T cells. Whereas blocking the PD-1/PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing-albeit not eliminating-both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8(+) T-cell exhaustion in patients with hematologic malignancies such as advanced AML.     

Trisomy 13 and myeloid malignancy — characteristic blast cell morphology: a United Kingdom Cancer Cytogenetics Group survey

We retrospectively report data on 28 patients with haematological malignancy and trisomy 13 (25 cases) or tetrasomy 13 (three cases) as the primary acquired cytogenetic change. Peripheral blood and/or bone marrow morphology was reviewed in 25/28 cases and the final diagnosis was as follows: AML M0 (11), AML M1 (6), AML M2 (2), AML M4 (2), AML M5b (1), AML M6 (1), RAEB-t (3), RAEB (1), RA (1). All three cases with tetrasomy 13 had AML M0. Characteristic small hand-mirror blasts with cytoplasmic blebs and tails and scanty small granules were seen in 13/25 cases and 18/25 cases had small blasts which could easily be mistaken for lymphoblasts. Trilineage dysplasia was present in 8/28 cases. Median patient survival was 3 months. We conclude that trisomy 13 is particularly associated with acute myeloid leukaemia with minimal differentiation (AML M0), often has distinctive morphological features, and has a poor prognosis.     

Widening plasty of the anterior cusp in rheumatic mitral insufficiency

The mechanism of insufficiency in rheumatic valve disease includes 1. annulus dilatation and 2. restricted leaflet motion. Aiming at improving the treatment of restriction, augmentation of the anterior mitral leaflet (AML) was achieved with a piece of autologous pericardium. METHODS: between January 1995 and December 1999, out of 274 patients refered for rheumatic mitral disease, 143 patients underwent a repair (52%), 81% of them had pure regurgitation with no stenosis. Ring annuloplasty was performed in all cases. Two techniques used for treating the restrictive componant of the regurgitation were compared in two consecutive cohort of patients: no AML augmentation (n=62) and AML augmentation (n=81). Mean age was 42 + 3 years and all preoperative variables were comparable except for the incidence of redo patients who all underwent AML extension. RESULTS: in hospital mortality was 0.7% (n=1 with AML extension) and there was one early reoperation for a pericardial patch dehiscence. After a mean follow-up of 3.2 years, there was one sudden death (no AML extension). The reoperation rate was lower with (2.5%) than without (12.9%) AML augmentation (p<0.05). Echographic study showed a lower incidence of recurrency of mitral insufficiency when AML augmentation had been performed (grade 2: 9% and grade 3: 3%) as compared to no AML augmentation (grade 2: 35% and grade 3: 14%) (p<0.05). The mitral orifice area was larger (AML augmentation: 2.2 + 0.3 cm2 vs no AML augmentation: 1.8 + 0.4 cm2). CONCLUSION: ring annuloplasty alone failed to correct rheumatic mitral insufficiency in all cases. AML augmentation improved the quality of the repair and decreased the risk of reoperation.     

IDH2 mutations are frequent in Chinese patients with acute myeloid leukemia and associated with NPM1 mutations and FAB-M2 subtype

Introduction: Gene mutations play an important role in acute myeloid leukemia (AML) pathogenesis. Several genes have been identified in AML, such as FLT3, KIT, NPM1, and JAK2. This study investigated the frequency of novel mutations in IDH1 (amino acid R132) and IDH2 (R140 and R172) and analyzed their impact on disease biology and interaction with other mutations in Chinese patients with de novo AML. Methods: A total of 195 patients were screened for mutations in the IDH1, IDH2, JAK2 V617F, NPM1, FLT3, and KIT genes, using polymerase chain reaction (PCR)-based and direct sequencing assays. Results: IDH mutations occurred at a considerable frequency of 15.89% in Chinese AML cases; IDH2 R140Q was the most frequent genetic alteration and was associated with older age, normal karyotype, and French-American-British classification M2 at diagnosis. There was a strong association of IDH2 mutation with NPM1 mutations and a trend with FLT3-internal-tandem duplication. Conclusion: IDH mutations may be a novel genetic marker in cytogenetically normal AML and may cooperate in leukemogenesis.     

CYP1A1*2B (Val) allele is overrepresented in a subgroup of acute myeloid leukemia patients with poor-risk karyotype associated with NRAS mutation,but not associated with FLT3 internal tandem duplication

The etiology of acute myeloid leukemia (AML) is largely unknown. Biologic and epidemiologic data implicate exogenous toxicants, including cytotoxic drugs, benzene, radiation, and cigarette smoking. Allelic variation in genes encoding enzymes such as NADP(H) quinone oxidoreductase (NQO1) and glutathione S-transferase T1 (GSTT1) that metabolize environmental toxicants predispose to subtypes of AML, including therapy-related AML. We assayed NRAS oncogene mutation and FLT3 internal tandem duplication in 447 AML patients with an abnormal karyotype treated in Medical Research Council (MRC) AML clinical trials. Functional allelic variant frequencies in genes encoding carcinogen-metabolizing enzymes GSTT1, GSTM1, CYP1A1, CYP2D6, CYP2C19, SULT1A1, and NQO1 were previously determined for this cohort. FLT3 internal tandem duplication (ITD) frequency was 17%, and NRAS mutation 12% for the entire cohort. The 2 mutations were found together in only 4 patients. No association was found between enzyme allelic variant frequencies and the presence of FLT3 ITD for the entire cohort or within cytogenetic subgroups. CYP1A1*2B (Val) high-inducibility variant allele was overrepresented in patients with NRAS mutation compared with no mutation, for (1) the entire AML cohort (n = 8/53 vs 26/371; odds ratio [OR] = 2.36; 95% confidence interval [CI] 1.01-5.53) and (2) the poor-risk karyotype group (n = 6/14 vs 4/89; OR = 15.94; 95% CI 3.71-68.52) comprising patients with partial/complete deletion of chromosome 5 or 7, or abnormalities of chromosome 3. The CYP1A1*2B allele may predispose to the development of these subgroups of AML by augmented phase 1 metabolism to highly reactive intermediates of CYP1A1 substrates, including polycyclic aromatic hydrocarbons, or by generation of oxidative stress as a metabolic by-product.     

Pharmacologic inhibition of CDK4/6: mechanistic evidence for selective activity or acquired resistance in acute myeloid leukemia

Entry into the cell cycle is mediated by cyclin-dependent kinase 4/6 (CDK4/6) activation, followed by CDK2 activation. We found that pharmacologic inhibition of the Flt3 internal tandem duplication (ITD), a mutated receptor tyrosine kinase commonly found in patients with acute myelogenous leukemia (AML), led to the down-regulation of cyclin D2 and D3 followed by retinoblastoma protein (pRb) dephosphorylation and G(1) cell-cycle arrest. This implicated the D-cyclin-CDK4/6 complex as a downstream effector of Flt3 ITD signaling. Indeed, single-agent PD0332991, a selective CDK4/6 inhibitor, caused sustained cell-cycle arrest in Flt3 ITD AML cell lines and prolonged survival in an in vivo model of Flt3 ITD AML. PD0332991 caused an initial cell-cycle arrest in well-established Flt3 wild-type (wt) AML cell lines, but this was overcome by down-regulation of p27(Kip) and reactivation of CDK2. This acquired resistance was not observed in a Flt3 ITD and a Flt3 wt sample from a patient with primary AML. In summary, the mechanism of cell-cycle arrest after treatment of Flt3 ITD AML with a Flt3 inhibitor involves down-regulation of cyclin D2 and D3. As such, CDK4/6 can be a therapeutic target in Flt3 ITD AML but also in primary Flt3 wt AML. Finally, acquired resistance to CDK4/6 inhibition can arise through activation CDK2.     

Effector mechanisms of sunitinib-induced G1 cell cycle arrest, differentiation, and apoptosis in human acute myeloid leukaemia HL60 and KG-1 cells

Acute myeloid leukaemia (AML) is a heterogeneous disease with dismal outcome. Sunitinib is an orally active inhibitor of multiple tyrosine kinase receptors approved for renal cell carcinoma and gastrointestinal stromal tumour that has also been studied for AML in several clinical trials. However, the precise mechanism of sunitinib action against AML remains unclear and requires further investigation. For this purpose, this study was conducted using human AML cell lines (HL60 and KG-1) and AML patients’ mononucleated cells. Sunitinib induced G1 phase arrest associated with decreased cyclin D1, cyclin D3, and cyclin-dependent kinase (Cdk)2 and increased p27^Kip1, pRb1, and p130/Rb2 expression and phosphorylated activation of protein kinase C alpha and beta (PKCα/β). Selective PKCα/β inhibitor treatment abolished sunitinib-elicited AML differentiation, suggesting that PKCα/β may underlie sunitinib-induced monocytic differentiation. Furthermore, sunitinib increased pro-apoptotic molecule expression (Bax, Bak, PUMA, Fas, FasL, DR4, and DR5) and decreased anti-apoptotic molecule expression (Bcl-2 and Mcl-1), resulting in caspase-2, caspase-3, caspase-8, and caspase-9 activation and both death receptor and mitochondria-dependent apoptosis. Taken together, these findings provide evidence that sunitinib targets AML cells through both differentiation and apoptosis pathways. More clinical studies are urgently needed to demonstrate its optimal clinical applications in AML.     

Involvement of the AML1 gene in the t(3;21) in therapy-related leukemia and in chronic myeloid leukemia in blast crisis

A nonrandom translocation between chromosomes 3 and 21, t(3;21)(q26.2;q22) has been detected in patients with a myelodysplastic syndrome or acute myeloid leukemia after treatment (t-MDS/t-AML) for a primary malignant disease and in chronic myelogenous leukemia in blast crisis (CML-BC). In these patients, the breakpoint on chromosome 21 is at band 21q22. This band is also involved in the t(8;21)(q22;q22) detected in 40% of the patients with acute myeloid leukemia subtype M2 (AML-M2) de novo who have an abnormal karyotype. In the t(8;21), the AML1 gene is the site of the breakpoint on chromosome 21. The AML1 gene is transcribed from telomere to centromere, and in the t(8;21) the 5' part of AML1 is fused to the ETO gene on chromosome 8 to produce the chimeric AML1/ETO on the der(8) chromosome. We found that AML1 is also rearranged in two t-AML patients and in one CML-BC patient with the t(3;21), but the breakpoints are approximately 40 to 60 kb downstream to those of AML-M2 patients. This region contains at least one additional exon of AML1, as determined by using an AML1 cDNA as a probe in Southern blot analysis. The t(3;21) breakpoints for the remaining patients could not be determined because, by fluorescence in situ hybridization analysis, the breaks are outside of the region covered by the available probes.     

Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H- TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.     

Autoimmune diseases,infections, use of antibiotics and the risk of acute myeloid leukaemia: a national population‐based case‐control study

Previous studies reported increased risk of acute myeloid leukaemia (AML) in individuals with inflammatory conditions. However, it is unclear whether this association is explained by preceding cytotoxic therapy or haematological diseases. We conducted a nationwide case‐control study that included 3053 AML patients, diagnosed in Denmark between 2000 and 2013, and 30 530 sex‐ and age‐matched population controls. We retrieved information on autoimmune disease, infections, and use of antibiotics and computed odds ratios for AML (conditional logistic regression). Results were stratified by AML type, sex, and age. Autoimmune diseases were associated with an overall increased risk of AML {odds ratio [OR] 1·3 [95% confidence interval (CI) = 1·1–1·5]}. However, the risk was confined to patients with previous haematological disease or cytotoxic therapy exposure [secondary/therapy‐related AML (sAML/tAML0) OR 2·0 (95% CI = 1·6–2·6)] and not de novo AML [OR 1·1 (95% CI = 0·9–1·3)]. Similarly, any prior infection requiring hospitalization was associated with a higher risk of AML [OR 1·3 (95% CI = 1·1–1·4)]. Again, this association was evident for sAML/tAML [OR 1·8 (95% CI = 1·5–2·2)], and not de novo AML [OR 1·1 (95% CI = 1·0–1·2)]. In conclusion, autoimmune diseases and infections were associated with an increased AML risk only in subjects with prior haematological disease and/or cytotoxic treatment. These observations suggest, that inflammation plays – if any – a minor role for the development of de novo AML.     

DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.