Variable cytotoxicity of diphtheria toxin 388-granulocyte-macrophage colony-stimulating factor fusion protein for acute myelogenous leukemia stem cells

In this study, the utility of DT388-granulocyte-macrophage colony-stimulating factor (GM-CSF) for the ex vivo purging and direct administration to patients with acute myeloid leukemia (AML) is tested using clonogenic assays, long-term cultures (LTC), and NOD/SCID mice as assays for leukemic progenitors.We compare the ability of 24-hour exposure to 0.3 microg/mL (4 nM) DT388-GM-CSF to kill AML colony forming cells (CFC) and the more primitive AML progenitors detected after 6 weeks in stromal cocultures (AML LTC-initiating cells or AML LTC-IC) and after 8 weeks in NOD/SCID mice.AML samples (n = 10), expressing a mean of 35 to 1466 GM-CSF receptors/blast, showed mean (range) percent kills of AML CFC and LTC-IC of 61 (17-98) and 46 (0-94) respectively with a direct correlation (r = 0.69) between the % kills detected in the in vitro assays. Among 5 evaluable samples the percent reduction in AML cell engraftment in NOD/SCID marrow following ex vivo DT388-GM-CSF treatment varied from 38% to 100%. 40% to 56% of normal bone marrow CFC and 31% to 48% of normal LTC-IC survived the same ex vivo treatment (n = 3). In subsequent experiments, NOD/SCID mice received AML blast cell injections intravenously followed in 24 hours by 1.5 microg DT388-GM-CSF daily intraperitoneally for 5 days. A reduction of marrow blast cells was seen with 7 of 9 samples tested 4 to 12 weeks post one course of toxin. Repeating the 5-day course of toxin 2 or 3 times at 4-week intervals did not improve the response, while delaying administration until 4 to 8 weeks post AML cell injection reduced the toxin's effectiveness (n = 5).This fusion toxin may prove useful for in vitro purging of stem cell harvests from selected AML patients and for direct administration to such patients.     

米托蒽醌为主的化疗方案对CD34+高表达急性髓细胞性白血病疗效观察

目的：探索米托蒽醌(MTZ)在急性髓细胞性白血病(AML)化疗中的作用特点，提高AML的疗效和无病生存率(FDS)。方法：80例免疫分型中有CD34^ 抗原高表达的AML，随机选择(MA／MAE、DA／DAE和HA／HAE)方案，联合化疗1～2个疗程后分别比较CR率、骨髓抑制及其它毒副作用；同时对白血病细胞进行体外药物杀伤效应实验，分别比较MTZ、柔红霉素(DNR)、高三尖杉酯碱(HHT)对白血病细胞不同分化阶段的抑制作用。结果：CD34抗原高表达的AML中，1～2个疗程CR率，以MA／MAE方案最高。为80．0％(24／30)．白血病细胞体外药物杀伤实验显示，MTZ对CD34^ 高表达的AML的抑制显著高于DNR和HHT。结论：MTZ具有较强的抗AML活性，临床骨髓抑制明显上述特点可能与其主要作用于AML白血病细胞的分化较早阶段有关。     

CAG-GO therapy for patients with relapsed or primary refractory CD33-positive acute myelogenous leukemia

We previously tested a less toxic CAG regimen consisting of low-dose cytarabine, aclarubicin and granulocyte-colony stimulating factor for the treatment of patients with relapsed or refractory myeloid malignancies or elderly patients with untreated ones, obtaining a satisfactory complete remission rate of 62%. Gemtuzumab ozogamicin, an anti-CD33 monoclonal antibody conjugated to calicheamicin, has recently been approved as a single agent in Japan for the treatment of relapsed/refractory CD33-positive acute myelogenous leukemia (9 mg/m(2) on days1 and 15). Complete remission rate was reported as 30% in a phase 2 trial in Japan. In this study, effectiveness and safety of combining dose-attenuated gemtuzumab ozogamicin (3 mg/m(2) on day5) and original CAG regimen were assessed in nine patients with relapsed/refractory CD33-positive acute myelogenous leukemia and a median age of 70 years. Rate of complete remission with or without platelet recovery was 44% (4/9). The median duration of complete remission and overall survival were 5.5 and 16 months, respectively. Reversible myelosuppression and liver toxicity were the main adverse events, but no regimen-related death was recorded. Although only a small number of cases were included in this preliminary study, this CAG-GO regimen was found to be feasible and useful even in high-risk relapsed or refractory patients.     

Sensitivity of human acute myeloid leukaemia to diphtheria toxin-GM-CSF fusion protein

The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM-CSF-diphtheria toxin fusion protein (DT-GM-CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G-CSF, huIL-3 and huSCF. DNA synthesis was maximally inhibited after 48 h exposure to DT-GM-CSF. Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT-GM-CSF, with 50% inhibition of DNA synthesis (ID₅₀) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT-GM-CSF (ID₅₀  99 ng/ml). From the 15 samples which showed autonomous proliferation, 13 were sensitive to inhibition of proliferation by DT-GM-CSF. The level of GM-CSF receptor (GM-CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT-GM-CSF was specifically mediated by the GM-CSFR, no correlation was found between the level of expression of the GM-CSFR alpha or beta subunit and the sensitivity for DT-GM-CSF. These in vitro studies show that the DT-GM-CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.     

急性白血病患者调节性T细胞的检测及临床意义

目的:探讨急性白血病患者外周血中CD4+CD25+调节性T细胞(Treg)的变化及意义。方法:采用流式细胞技术及ELISA方法检测56例急性白血病患者外周血中CD4+CD25+ Treg、IL-10的变化,并与健康对照组进行比较。结果:急性白血病患者外周血中CD4+CD25+/CD4+CD25high Treg占(23.85±6.85)%及(5.21±1.20)%,明显高于健康对照组(P0.01);56例急性白血病患者血清中IL-10水平为(74.4±21.2)pg/ml,明显高于正常对照组(24.6±8.7)pg/ml(P0.01),与CD4+CD25+/CD4+CD25 high Treg存在正相关(r=0.253/r=0.511,P0.05)。结论:急性白血病患者外周血CD4+CD25+/CD4+CD25 high Treg水平明显升高,与急性白血病患者免疫逃逸密切相关。     

Polyclonal normal hematopoietic progenitors in patients with acute myeloid leukemia

OBJECTIVE: To study the clonality of cytogenetically normal progenitors detected in the peripheral blood (PB) of acute myeloid leukemia (AML) patients. METHODS: Five female patients with cytogenetically abnormal, newly-diagnosed AML who were heterozygous for informative alleles of the androgen receptor (AR) gene were studied using the human androgen receptor allele (HUMARA) assay. RESULTS: PB mononuclear cells and bone marrow (BM) fibroblasts from these patients were monoclonal and polyclonal, respectively. Both cytogenetically normal and abnormal colony-forming cells (CFC) were detected from 3 AML samples and the HUMARA assay determined that most of these CFC were part of the leukemic clone. The fourth sample generated colonies that were 100% normal by cytogenetics and polyclonal by HUMARA. In contrast, 5-week-old long-term culture (LTC)-derived colonies were 100% cytogenetically normal by FISH and polyclonal by HUMARA in 4 of the 5 samples. The fifth sample, which showed a small number of karyotypically abnormal LTC-derived colonies, nevertheless showed amplification of the "leukemia-associated" AR allele in 46/50 LTC-derived colonies as well as all 40 directly clonogenic cells tested.CONCLUSIONS: Thus in 4 of 5 AML samples tested, both cytogenetics and the HUMARA assay indicate that a substantial number of normal, polyclonal hematopoietic progenitors often persist in AML PB at diagnosis in spite of the predominance of malignant blasts and the severe cytopenias of normal mature blood cells that are typically seen clinically.     

Circulating T cells in patients with untreated acute myelogenous leukemia are heterogeneous and can be activated through the CD3/TCR complex

T lymphocyte defects may contribute to the immune insufficiency seen in acute myelogenous leukemia (AML). We therefore characterized the T cell system for untreated AML patients. T lymphocyte subsets were analyzed by flow cytometry for 45 AML patients. The in vitro interferon-gamma (IFNgamma) release in response to stimulation with anti-CD3 plus anti-CD28 in the presence of autologous AML cells was examined for 31 patients. The majority of circulating lymphocytes were CD3(+)T cells, and CD19(+)B cells usually constituted < 10% of the lymphocytes. Most T cells expressed the alphabeta T cell receptor (TCRalphabeta(+)), and only a minority of the cells was TCRgammadelta(+). Both CD4(+) and CD8(+)T cells were detected, the CD4:CD8 ratio showed a wide variation but was generally >1.0. The majority of CD4(+) and CD8(+)T cells were CD45RA(+) cells. The T cells could be stimulated to release IFNgamma in response to anti-CD3 plus anti-CD28 ligation even in the presence of excess autologous AML blasts, and for a subset of patients (6 of 27) these IFNgamma levels could be further increased by the novel protein kinase C (PKC) agonist PEP005. In conclusion, circulating T cells in patients with untreated AML are mainly CD4(+) or CD8(+) TCRalphabeta(+); both CD45RA(+) and CD45R0(+) can be detected, and these cells can be activated through the CD3/TCR complex even in the presence of excess AML cells. For a subset of patients T cell responsiveness can be further increased by targeting PKC and these data therefore suggest that T cell function is not inhibited in AML patients.     

Life-threatening hypophosphatemia in a patient with acute myelogenous leukemia

A patient with acute myelogenous leukemia developed severe hypophosphatemia manifesting by extreme weakness, confusion, loss of sphincter control, nuchal rigidity, hyperesthesia, hemolysis, congestive heart failure and liver dysfunction. The possible causes for this condition were starvation, parenteral glucose and saline administration, sepsis, hypokalemia and treatment with acetazolamide. A dramatic improvement was noted following phosphate administration.     

Heat shock protein 90 inhibition sensitizes acute myelogenous leukemia cells to cytarabine

Previous studies demonstrated that ataxia telangiectasia mutated- and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.     

Acute myeloid leukemia cells in G0 phase of the cell cycle that are unresponsive to conventional chemotherapy are sensitive to treatment with granulocyte-macrophage colony-stimulating factor/diphtheria toxin fusion proteins

OBJECTIVE: Unresponsiveness to chemotherapy is a major problem in the treatment of leukemia, which can be caused by unresponsiveness of noncycling cells to cell cycle-dependent cytotoxic agents. Targeted toxins consisting of a targeting and activating cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and diphtheria toxin (DT) can be used to overcome this kind of resistance of leukemic cells. In this study we manipulated the cell cycle and proliferative status of leukemic cells, explored the effect on sensitivity to DT, and determined the ability of DT388GMCSF fusion proteins to activate and subsequently kill leukemic cells. MATERIALS AND METHODS: We used the GM-CSF-dependent myeloid leukemic cell line AML-193 as a model. GM-CSF or granulocyte colony-stimulating factor (G-CSF) was used to manipulate the cell cycle and proliferative state of AML-193 cells. Cell death was quantified by 51Cr release assays. The results obtained in the AML-193 cell line model were confirmed using primary leukemic blasts. RESULTS: Similar to treatment with chemotherapy and immunotherapy, leukemic cells in resting G0 phase were relatively resistant to DT-induced cell death. Synchronized recruitment of leukemic cells into activated phases of the cell cycle by low concentrations of GM-CSF or G-CSF resulted in significant increased DT sensitivity. DT388GMCSF fusion proteins specifically targeted GM-CSF receptor-expressing cells, resulting in recruitment of leukemic cells from G0 phase of the cell cycle and subsequent kill of these cells. CONCLUSION: Leukemic cells in G0 phase, which are resistant to conventional chemotherapy, Fas-induced immunotherapy, and DT alone, can be synchronically activated and subsequently killed by DT388GMCSF fusion proteins.     

Granulocytic sarcoma of the ovary in patients with acute myelogenous leukemia

Two cases of granulocytic sarcoma of the ovary found at the time of relapse of acute myelogenous leukemia (AML) are presented. Almost 5 years after the initial diagnosis of AML, both cases presented ovarian tumors which were discovered to be myeloblastoma of the ovary on laparotomy in one case and at the time of autopsy in the other. Evidence of hematological relapse followed the presentation of the ovarian tumor within a month. Both patients were treated with surgery and/or chemotherapy.     

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.     

Conservative treatment for patients over 80 years with acute myelogenous leukemia

In order to evaluate the best treatment of very elderly patients with AML, we have retrospectively analyzed 60 cases of patients aged more than 80 years, with a diagnosis of AML and observed from January 1988 to December 1998. Six of these patients were subsequently referred to other centers; of the remaining 54 patients, 20 (37%) received only supportive care, whereas 34 (63%) required palliative chemotherapy to control leukocytosis, after a median time from diagnosis of 9 days (range 0-253). Median overall survival was 13 weeks (range 1-105): 21 (39%) and 6 (11%) patients survived more than 6 and 12 months, respectively. Twenty-eight patients (51.8%) died from progressive disease, 19 (35.1%) died from AML-related or unrelated causes in the phase of stable disease, while in 7 patients the cause of death was unknown. In univariate analysis, PS > 2 and WBC > 50 x 10(9)/L had an adverse prognostic significance on survival. Our results, as compared with those reported in the literature for patients over 80 years treated with intensive chemotherapy, support the idea that intensive chemotherapy is usually not indicated in very elderly patients with AML, and that conservative treatment and the primary strategy of "watch-and-wait" presently seems to be the best choice.     

Beneficial effects of hepatitis in patients with acute myelogenous leukemia.

Of 50 consecutive patients admitted with acute myelogenous leukemia, 30 developed complete remissions on antileukemic therapy. Nineteen of the 30 repeatedly had elevated serum glutamic oxalacetic transaminase (SGOT) concentrations 3 to 14 weeks after the start of therapy. Patients with SGOT elevations had a significantly greater chance of remission and a longer survival (76 +/- 11 weeks) than those with normal SGOT levels (39 +/- 5 weeks), suggesting that hepatitis may have a beneficial effect in acute myelogenous leukemia. The hepatitis was mild in all patients. Review of patients at this institution alive 2 years after the diagnosis of acute myelogenous leukemia showed that they repeatedly had elevated SGOT levels. We believe that most had non-A, non-B post-transfusion hepatitis, which may have a beneficial effect on the leukemia or serve as an indicator of patients who have greater immunocompetence and thus a better prognosis.     

Treatment of 61 patients with acute myelogenous leukemia at first relapse

Treatment results in the 61 adult patients with AML in first relapse were analyzed to establish a better strategy for this group of patients. These patients received reinduction chemotherapy during 1979-1988. Complete remission (CR) was obtained in 57.4% of the cases, and the probability of survival and remaining in CR at five years was 11.9% and 17.9%, respectively. The longer duration of initial remission was favorable factor for achieving second CR. Type of reinduction regimens which were different from those used in the initial induction phase did not influence the second CR rate. The use of different consolidation regimen appeared to favorably affect the survival and probability of remaining in CR.     

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.