t(8;21)急性髓系白血病患者MICM分型及预后的回顾性分析

目的 分析t(8;21)急性髓系白血病(AML)患者的细胞形态学、免疫表型、遗传学、分子生物学(MICM)分型及临床治疗疗效.方法 运用瑞特染色法、FAB细胞形态分类标准、流式细胞术(FCM)直接免疫荧光标记技术、遗传学染色体吉姆萨显带技术及RT-PCR技术对70例确认有t(8;21)与AML1-ETO融合基因双阳性的AML患者及70例正常染色体核型的AML患者进行分析和比较.结果 70例t(8;21)AML患者中M11例,M2 64例,M4 3例,无法分型的急性白血病(AL)2例;免疫表型分析发现CD13、CD33、CD34、CD117高表达,40%表达CD19,11%表达CD15,10%表达CD11b,7%表达CD7;遗传学显示50%的t(8;21)AML患者有附加染色体异常,主要为性染色体丢失、9q-及超二倍体;RT-PCR检测AML1-ETO融合基因100%阳性.CD+19t(8;21)AML患者完全缓解(CR)率72%,CD+19伴CD+7t(8;21)AML患者CR率为0,正常核型CR率31%.结论 t(8;21)AML患者主要在M2中集中出现,附加染色体异常较多见.CD19表达较高,而CD7表达极低,CD34、CD117高表达,这些抗原的表达可能与核型密切相关.CD+19是预后良好的指标,但同时出现CD+7,则预后不良.     

Multiple sclerosis: use of MRI in evaluating new therapies

11q23 translocations (t(11q23)) are recurring cytogenetic abnormalities in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia, involving the same gene, ALL1 (or MLL). Mixed lineage antigen expression has been reported in these leukemias, but its frequency and clinical significance are unknown. We immunophenotyped leukemia cells from 19 adult de novo AML patients with t(11q23) by multiparameter flow cytometry. Translocations included t(6;11)(q27;q23), t(9;11)(p22;q23), t(9;11;19)(p22;q23;q13.3), t(2;11)(11;17)(q37;q11q23;q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) and t(11;22)(q23;q11). FAB types were M4 and M5. The committed stem cell and myeloid antigens HLADr, CD4dim, CD11b, CD13, CD15, CD32, CD33, CD38 and CD64 were each expressed in 80-100% of cases, and the early stem cell and lymphoid antigens CD34, CD56, CD3, CD2 and CD7 in 42, 39, 16, 5 and 5%, respectively. Antigen expression frequencies did not differ from those in 443 adequately karyotyped M4 and M5 cases without t(11q23). Fifteen patients (79%) attained complete remission (CR); median CR duration and survival were 10.0 and 15.1 months. CR duration and survival did not correlate with antigen expression. In particular, patients with t(9;11) survived longer than those with other t(11q23) (median not reached vs 7.6 months; P = 0.048), but antigen expression did not differ in the two groups. Thus frequencies of lymphoid antigen expression are similar in AML with t(11q23) and in other FAB M4 and M5 cases, treatment outcome does not differ in t(11q23) cases with and without lymphoid antigen expression, and better outcome of patients with t(9;11) compared to other t(11q23) does not correlate with differences in antigen expression. Mixed lineage antigen expression is not a distinctive feature of AML with t(11q23).     

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.     

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23)

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.     

应用LSI9q34探针及ES/DCDF探针检测慢性粒细胞白血病患者der(9)缺失的研究

目的 研究慢性粒细胞性白血病(CML)患者衍生9号染色体[der(9)]部分序列缺失情况,探讨LSI 9q34探针在检测der(9)缺失中的联合应用价值.方法 对52例CML患者采用不加任何刺激剂的骨髓进行24 h短期培养法制备染色体,吉姆萨显带进行核型分析.应用ES/DCDF探针及LSI 9q34探针对骨髓间期细胞进行荧光原位杂交,并检测der(9)部分序列缺失.结果 52例中经ES/DCDF探针检测全部为ber-abl融合基因阳性,其中12例患者伴有der(9)部分缺失,占23.0%,经LSI 9q34探针检测有11例患者伴有der(9)部分缺失.结论 LSI9q34探针在判断CML患者是否伴有der(9)缺失方面有较好的特异性.伴有缺失的患者疾病进展较快,预后较差,甲磺酸依马替尼治疗不能完全逆转其负性作用,建议所有bcr-abl融合基因阳性的CML患者均应行 LSI 9q34探针检测,以指导临床治疗.     

Functional expression of Fas (CD95) in acute myeloid leukemia cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy

Clinical studies of bone marrow transplantation (BMT) suggest that the immune system contributes to the eradication of acute myeloid leukemia (AML). A recent study also showed that the Fas (CD95/APO1) mediates apoptotic signal from cytotoxic T lymphocytes. Sixty-four patients with AML were studied for the expression of Fas in the context of CD34 and CD38 coexpression. The clinical relevance of Fas expression and function on AML was also investigated. Fas was expressed on 2% to 98% of AML cells (2% to 20% in 11 patients, 20% to 50% in 20 patients, 50% to 80% in 24 patients, and 80% to 98% in nine patients). Only 44.4% of patients with AML M1 (French-American-British [FAB] classification) were Fas+ (>/=20% of leukemia cells expressed Fas), whereas 89.1% of patients with AML M2, M3, M4, M5 were Fas+ (P < .01). Among 43 CD34+ patients (>/=20% leukemia cells were CD34+), 34 were Fas+, and 19 of 21 CD34- patients were Fas+ (P = NS). Thirteen cases were studied for their expression of Fas in the context of CD34 and CD38 using three-color analysis. Fas is expressed at a high level in the gated CD34+CD38+/- and CD34+CD38+ population. In 10 AML samples, Fas was expressed at a higher level in CD34+/CD38+ population than in CD34+/CD38+/- or CD34- cell populations. Fas-induced apoptosis by anti-Fas monoclonal antibody (MoAb) was determined by morphologic features and colorimetric DNA fragmentation assay. Induction of apoptosis was found in 14 of 24 cases. However, no statistically significant correlation was observed between Fas expression and induction of apoptosis. Leukemia colony-forming unit assays suggested that in some cases, Fas-induced apoptosis occurred in the clonogenic cell populations. Parameters such as laboratory and clinical data at initial diagnosis were correlated with Fas expression and only response to initial induction chemotherapy showed significant correlation with Fas expression (P < .05). We conclude that the majority of AML cells exhibit variable expression of Fas, and apoptosis could be induced by anti-Fas MoAb in some cases. Our results suggest the Fas-mediated apoptosis may be clinically relevant, whereas the issue of clonogenic leukemia cells and Fas expression needs further studies.     

Transjugular intrahepatic portosystemic shunt: a successful treatment for hepatopulmonary syndrome

ATP-binding cassette (ABC), ATP-dependent transporters are a large superfamily of proteins that include the multidrug resistance proteins, P-glycoprotein and MRP (multidrug resistance protein). The ARA (anthracycline resistance-associated) gene that codes for a putative member of the ABC transporters has recently been cloned and shown to have high sequence homology to the gene for MRP. We have previously shown MRP to be deleted in a subset of inv(16) leukemic patients. The deletion of MRP was associated with an improved patient survival compared with inv(16) patients who did not have such a deletion. In this study, the ARA gene is mapped to 16p13.1, in the same physical interval as the inv(16) short-arm breakpoint. It is shown to be situated proximal to both MYH11, the gene involved in the primary breakpoint on the short arm of the inv(16), and MRP. A YAC clone has been isolated containing both MRP and ARA. FISH analysis of metaphase chromosomes from inv(16) patients has established the gene order as telomere-MYH11-MRP-ARA-centromere and demonstrated that both ARA and MRP are deleted in a subgroup of the inv(16) leukemias. ARA and MRP are both shown to be expressed in normal hematopoietic precursors including CD34(+) cells. The mapping of ARA to this region and its homology to MRP raises questions about its potential role in the biology of the inv(16) leukemias.     

Myelomonocytic antigens in B-cell chronic lymphocytic leukemia

The clinical significance of myelomonocytic (MyMo) antigens in B-cell chronic lymphocytic leukemia (B-CLL) is unclear. We have analyzed the expression of MyMo antigens (CD13, CD14 (LeuM3, My4, Mo2), CD15, CD11b, CD11c, CD33 and CD68) on B-lymphocytes (CD19+) in 105 B-CLL patients and in 35 controls. A double direct staining technique and flow cytometric analysis was performed. The expression of MyMo antigens on the control group did not exceed 4% B-lymphocytes. A MyMo antigen was considered as positive when present in > or = 10% of B-lymphocytes. Among the B-CLL patients, 28 (26.7%) were positive for CD11c, 21 (20.0%) for CD11b, nine (8.6%) for CD15, five (4.8%) for CD13, two (1.9%) for Mo2, and one (1.0%) for My4. No patient was positive for LeuM3, CD33 or CD68. CD11c was more frequently expressed in patients with a short lymphocyte doubling time (< 12 months) (P = 0.05) and CD11b in the group with a higher number of lymphoid areas involved (P = 0.02). No correlation was found between lymphoid morphology and MyMo antigen expression. Fourteen of the 80 patients at risk subsequently progressed to a more advanced stage. Multivariate analysis identified hemoglobin (P = 0.004) and CD11b positivity (P = 0.009) as independent variables for disease progression. Fifteen patients died during evolution. Seven out of the 21 CD11b positive patients and eight of the 84 CD11b negative patients died (LR: P = 0.02, BG: P = 0.05). In the multivariate analysis, only CD11b positivity (> or = 10%) added prognostic value to clinical stages.     

Clinicopathological features in HLA-DR-negative acute myeloid leukemia

From a series of 176 patients with acute myeloid leukemia (AML), we have identified 11 patients with HLA-DR-negative AML excluding acute promyelocytic leukemia. All patients showed not (15; 17) or consistent chromosomal abnormalities. According to the French-British-American criteria, seven, three, and one patients were classified into M1, M2, and M4, respectively. Blasts from these patients were CD33+, HLA-DR-, and lymphoid-antigen-. All patients entered complete remission after induction therapy. Of these, blasts from four patients had strikingly invaginated nuclear membrane and finely granular peroxidase activity. The phenotype of the blasts was CD33+, CD34-, CD2-, and HLA-DR-. All four patients showed hyperleukocytosis on initial presentation. One patient received all-trans retinoic acid at relapse, however, the drug did not induce the differentiation of the blasts. These four patients were suspected to have acute myeloid/natural killer (NK) leukemia because of the prominent morphological feature of the blasts and the initial hyperleukocytosis, although NK cell activity of the blasts was not examined. Since HLA-DR-negative AML is heterogeneous, it is necessary for identifying acute myeloid/ NK leukemia within the disorder.     

Potent antileukemic activity of the novel agents norsegoline and dibezine

We examined the effect of norsegoline, a natural marine product, and dibezine, a synthetic product, on the survival of human myeloid progenitor cells [colony-forming unit-cells (CFU-C)] from normal individuals and from 10 patients with Philadelphia-positive chronic myelogenous leukemia (CML) in chronic phase and blastic crisis. We compared their effect to the effect of IFN-alpha. Norsegoline, dibezine, and IFN-alpha inhibited the proliferation of CFU-C in a dose-dependent manner. The number of CFU-C from bone marrow (BM) of five CML patients in chronic phase exposed for 16 h to norsegoline (10(-8)-10(-6)M), dibezine (10(-8)-10(-6)M), and IFN-alpha (500 units/ml) was found to be statistically lower (P < 0.05) than the number of CFU-C derived from normal individuals. A 16-h drug exposure of CD34(+) cells isolated from the peripheral blood of three CML patients in blastic crisis and from BM of two patients in chronic phase resulted in a marked inhibition in the ability of the cells to proliferate in liquid culture and a reduction in CFU-C content. Using the fluorescent in situ hybridization technique, we evaluated detection of the BCR/ABL fusion product in the CD34(+) cells. All five patients were 100% Philadelphia positive at diagnosis. BCR/ABL translocations were detected in 94.6 +/- 0.6% of cells following their growth in liquid culture for 7 days. Following exposure of CD34(+) cells to norsegoline, dibezine, or IFN-alpha, BCR/ABL fusion signals could be detected in 73 +/- 11%, 66.5 +/- 4. 7%, and 66.0 +/- 2.5% of cells from BM and 72.3 +/- 5%, 68.8 +/- 7%, and 60.6 +/- 6.8% of peripheral blood, respectively. Our data indicate that norsegoline and dibezine have in vitro an antileukemic effect against Philadelphia-positive cells and may be used in conjunction with currently available agents for ex vivo purging of BM and/or peripheral blood of CML patients in conjunction with autologous bone marrow transplantation.