Treatment of Acute Nonlymphocytic Leukemia by Concurrent Administration of N4-Behenoyl-1-{beta}-D-Arabinofuranosylcytosine, Aclacinomycin A, 6-Mercaptopurine and Prednisolone: A Pilot Study

N⁴-behenoyl-1-ß-d-arabinofuranosylcytosine (BH-AC),a newly synthesized derivative of cytosine arabinoside witha longer plasma half life and marked dose independency in itsanti-L1210 leukemia effect, and aclacinomycin A, a new anthracyclineantibiotic which is as effective as daunorubicin against L1210leukemia and reportedly induces no alopecia and is much lesscardiotoxic, were combined with 6-mercaptopurine and prednisolonein a pilot study of the effect on acute nonlymphocytic leukemiaof adults. The treatment schedule consisted of daily administrationof BH-AC, 116 to 234 mg/m² as a 2-hr intravenous infusion; aclacinomycinA, 12 to 16 mg/m² as a 30-min intravenous infusion; 6-mercaptopurine,100 mg/m² orally; and prednisolone, 40 mg/m² orally. A course of treatment was continued until the bone marrow becameseverely hypoplastic, not extending over 14 days. Eight patientswere entered in the study, one of whom was nonevaluable becauseof early death from renal failure. Six out of the seven evaluablepatients attained complete remission and the other good partialremission. Only one course of treatment was administered tofive of the patients who attained complete remission. It shouldbe noted that complete remission was obtained in two of threepatients not responding to treatment with daunorubicin, cytosinearabinoside, 6-mercaptopurine riboside and prednisolone in combination.The median duration of complete remission and survival were5 + (range: 4 + to 6 +) mo and 9 + (range: 6 + to 27+) mo, respectively. The most frequent side effects were malaise and gastrointestinalsymptoms, which were well tolerated. Flat T waves were notedin the electrocardiogram of one elderly patient; the changewas not clearly attributable to the drugs employed. Mild alopeciawas seen in two patients. This regimen was quite effective asa remission inducer in acute nonlymphocytic leukemia of adults,and deserves further study.     

Phase I and Preliminary Phase II Studies on Aclacinomycin A in Patients with Acute Leukemia

Eight patients with acute nonlymphocytic leukemia (ANLL) andfive patients with acute lymphocytic leukemia were treated withaclacinomycin A. It was given daily by one-hour infusion indoses ranging from 0.33 to 0.70 mg/kg for seven to 20 days withoutother antileukemic agents. Two patients with ANLL achieved completeremission and one with ANLL achieved partial remission. Itsmajor toxic effects were myelosuppression and gastrointestinalsymptoms.     

Comparative genotoxicity of 2,3'-O-cyclocytidine, {beta}-xylocytidine and 1-{beta}-D-arabinofuranosylcytosine in human tumor cell lines

We have Investigated the genotoxicity of two 3'-derivativesof cytidine, 2,3'-O-cyclocytidine (3'-cycloC) and ß-xylocytidine(xyloC), in human leukemia and solid tumor cell lines. Bothderivatives were found to be cytotoxic at micromolar concentrations.For example, in the alveolar tumor cell line A549 which wasincluded in all experiments as a reference, drug concentrationsrequired to induce 50% inhibition of cell growth (DM values)equalled 55 (iMfor 3'-cycloC and 80 µM for xyloC. Comparedwith the response of this reference cell line, none of the solidtumor cell lines tested—representing five different malignancies—displayedsignificant hypersensitivity to these drugs, while the acutelymphoblastic leukemia cell lines proved to be hypersensitive(range of D₅₀ values, 5–13 (µM). To gain insightinto the modes of cytotoxic action of xyloC and 3'-cycloC, wecompared the effect on DNA metabolism of these compounds withthat of 1-p-D-arabinofuranosylcytosine (araC), a potent inhibitorof semi-conservative DNA replication and long-patch excisionrepair. As seen with araC, the xylo compound strongly inhibitedboth DNA replicative synthesis and the repair of DNA damageinduced by UV light and ⁶⁰Co -radiation. In -irradiated A549cells, the extent of repair inhibition by 1 mM xyloC was 40%of that inhibited by araC, and concomitant exposure of the irradiatedcultures to xyloC plus araC gave rise to a synergistic response.Since araC was employed at a concentration (0.1 mM) which produceda maximal effect on DNA repair when applied alone, the observedsynergistic response implies that the mode of action of xyloCon DNA repair is different from that of araC. In contrast tothat observed with xyloC, 3'-cycloC proved to be a very weakinhibitor of DNA replication and repair, strongly suggestingthat the genotoxic action of the latter analog may be througha mechanism other than inhibition of DNA synthesis.     

Preliminary Phase II Study of Aclacinomycin A in Patients With Adult Acute Leukemia

Seventeen patients with adult acute leukemia in relapse weretreated with aclacinomycin A in 5-day courses given at a doseof 90 to 210 mg/m²/5 days as a daily bolus injection. Completeremission was achieved in patients treated with 130 or moremg/m²/5 days. Three out of seven patients with acute nonlymphocyticleukemia achieved complete remission lasting 15 mo, 14+ mo,and 2 mo. Of seven patients with acute lymphocytic leukemia,one achieved complete remission which lasted more than 11 mo.One patient with refractory anemia with an excess of blast cellsalso achieved complete remission. Neutropenia and thrombocytopeniaoccurred in all patients. The dose-limiting factors were gastrointestinalsymptoms and stomatitis, the latter occurring in three of theseven patients who received 200–210 mg/m²/5 days. Hyperbilirubinemiaassociated with antibiotic therapy was seen in four patients.Since aclacinomycin A has significant activity in acute nonlymphocyticleukemia, consideration should be given to exploring its usein combination with other active drugs.     

Aphidicolin and 1-{beta}-D-arabinofuranosylcytosine strongly inhibit transcriptionally active DNA repair in normal human fibroblasts

Both aphidicolin and 1-ß-D-arabinofuranosylcytosine(araC) inactivate DNA polymerases     

In situ analysis of transforming growth factor-{beta}s (TGF-{beta}1, TGF-{beta}2, TGF-{beta}3and TGF-3 type II receptor expression in malignant melanoma

We have analysed, by in situ hybridization, mRNA expressionof TGF-ß1, TGF-ß2, TGF-ß3, andof TGF-ß type II receptor in benign melanocytic naevi,primary melanomas, and in skin metastases of malignant melanomas.Our results show that melanoma progression correlates with overexpressionof TGF-ß. All skin metastases and most primary melanomasinvasive to Clark's level IV-V revealed specific TGF-ß2mRNA and protein expression. However, expression of this cytokinewas not observed in benign melanocytic lesions and was detectedonly in one of five early primary melanomas investigated. Someprimary melanomas and skin metastases also revealed specificTGF-ß1 mRNA signals although expression of this isoformwas not found in benign naevi. TGF-ß3 expression,which was only barely detectable in benign melanocytic lesions,was enhanced in some skin metastases. Interestingly, the epidermisoverlaying melanomas revealed lower levels of TGF-ß3mRNA expression than epidermis of healthy skin or epidermisadjacent to benign naevi, thereby suggesting that paracrinemechanisms between tumour cells and keratinocytes may influencemelanoma development. In primary melanomas TGF-ß typeII receptor mRNA signals were much more heterogeneously distributedwhen compared to benign melanocytic naevi, suggesting variabledegrees of TGF-ß resistance among melanoma cells withinindividual lesions. However, melanoma progression appeared notto be correlated with a complete loss of TGF-ß typeII receptor gene expression, since all skin metastases revealedclearly detectable although heterogeneous levels of TGF-ßtype II receptor mRNA expression.     

A Phase II Study of Continuous Infusion of Trimetrexate in Patients with Refractory Acute Leukemia

Trimetrexate, a second-generation folate antagonist, is a potent inhibitor of dihydrofolate reductase with a broader spectrum of activity and different mechanism of entry and intracellular accumulation than methotrexate. Six patients with refractory or relapsed acute leukemia were treated with a 5-day continuous infusion of trimetrexate of 8 mg/m² /day after an initial loading dose of 4 mg/m² to achieve a target plasma concentration of 0.2-0.5 μM. In 4 patients with peripheral blasts at study entry, transient decrease or disappearance of blasts was observed, although no decrease of bone marrow blasts occurred. Mucositis was dose-limiting and severe in 4 patients. Neutrophil and platelet nadirs occurred on day 5-12 postinfusion. Because of dose-limiting mucositis, this dose schedule of trimetrexate is not recommended for further studies in refractory acute leukemia. However, other dose schedules (24- to 72-hr infusions) and its use as a modulating agent with thiopurines or leucovorin in patients that are resistant to methotrexate should be explored.     

Inhibitory Anti-Tumor Effects of Interleukin-4 on Philadelphia Chromosome-Positive Acute Lymphocytic Leukemia and Other Hematopoietic Malignancies

IL-4 has been shown to possess a broader spectrum of biological activities. Recently, anti-tumor activities of IL-4 on malignant tumors including hematopoietic tumors has been revealed in vitro or in vivo. We investigated the effect of recombinant human (rhIL-4) on the in vitro growth of human leukemia cells and demonstrated the inhibitory anti-tumor activity of rhIL-4 on Ph¹-positive ALL cells in association with the decreased activity of cellular tyrosine kinase. This finding suggests that the clinical evaluation of rhIL-4 may offer promising therapeutic possibility for patients with Ph¹-positive ALL. In this paper, we presented the IL-4-dependent inhibition of Ph¹-positive ALL cells and reviewed implications for mechanism of IL-4-dependent inhibition and anti-tumor activities of IL-4.     

Engagement of I-branching {beta}-1, 6-N-acetylglucosaminyltransferase 2 in breast cancer metastasis and TGF-{beta} signaling

In this study, we have showed that GCNT2, a gene-encoding glucosaminyl (N-acetyl) transferase 2, I-branching enzyme, is overexpressed in highly metastatic breast cancer cell lines of human and mouse origin and basal-like breast tumor samples. GCNT2 expression is also significantly correlated to the metastatic phenotype in breast tumor samples. Functional studies showed that ectopic expression of GCNT2 enhances cell detachment, adhesion to endothelial cells, cell migration and invasion in vitro, and lung metastasis of breast cancer cells in vivo. Knockdown of GCNT2 expression decreases cell migration and invasion in vitro and lung metastasis in vivo. We have further shown the involvement of GCNT2 in the epithelial-to-mesenchymal transition (EMT). Specifically, the expression of E-cadherin is significantly changed upon GCNT2 expression at the protein level but not at the RNA level. Moreover, we have shown that GCNT2 is a direct target of the TGF-β-smad pathway and that change in GCNT2 expression modulates EMT induced by TGF-β1 treatment. Finally, we have shown that diminution of the glycosyltransferase activity of I-branching β-1, 6-N-acetylglucosaminyl transferase 2 (GCNT2) abrogates its cell migration and invasion-promoting function and synergistic effect with TGF-β to induce EMT. Our study for the first time showed that GCNT2 is a novel gene contributing to breast cancer metastasis with preferential expression in basal-like breast cancer. Moreover, we discovered that involvement of GCNT2 in EMT and TGF-β signaling, and further glycosylation modification of E-cadherin by GCNT2, are the underlying integrative mechanisms for breast cancer metastasis, implying that blocking TGF-β/GCNT2 signaling is a promising approach for targeting metastatic breast cancer.     

Comparative mutagenicity of 4-(carbethoxynitrosamino)-4-(3-pyridyl)butanal and 4-(carbethoxynitrosamino)-1-(3-pyridy1)-1-butanone, model compounds for {alpha}-hydroxylation of N'-nitrosonornicotine

4-(Carbethoxynitrosamino)-1-(3-pyridy1)butanal, a stable precursorto the putative diazohydroxide formed by 5'-hyd-roxylation ofthe tobacco-specific nitrosamine, N'-nitrosonornicotine, wassynthesized in six steps from nicotinaldehyde. Its mutagenicitytoward S. typhimurium was compared to that of 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone,a precursor to the diazohydroxide formed by 2'-hydroxylationof N'-nitrosonornicotine. At equimolar doses, 4-(carbethoxy-nitrosamino)-1-(3-pyridy)-1-butanonewas a potent mutageon, but 4-(carbethoxynitrosamino)-1-(3-pyridy)butanal was inactive toward strains TA 100 and TA 1535. Theresults of this study indicate that the putative diazohydroxideformed by 2'-hydroxylation of N'-nitrosonornocotine has higherinherent mutagemicity toward S. typhimurinum than does the correspondingdiazohydroxide formed by 5'-hydroxylation.     

Effects of {alpha}-deuterium substitution on the mutagenicity of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenictobacco specific nitrosamine, can be converted to electrophilicdiazohydroxide intermediates by metabolic hydroxylalion of eitherthe methylene carbon (carbon 4) or the methyl carbon attachedto the nitrosamine group. To investigate the relative importanceof these two processes in NNK mutagenesis, we synthesized 4,4-dideutero-4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone([4,4,-D₂]NNK)and 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone([CD₃] NNK), and evaluated their mutagenic activities in Salmonellatyphimurium tester strains. In the presence of Aroclor inducedrat liver 9000 g supernatant, NNK and [4,4-D₂]NNK had comparablemutagenic activities towards S. typhimurium TA 1535 and TA 100,but [CD₃]NNK was inactive in both strains. These results suggestthat hydroxylation of the methyl group of NNK is more importantthan hydroxylation of carbon 4 in its activation to a mutagen.To test the inherent mutagenicity of 4-oxo-4-(3-pyridyl)butyldiazohydroxideand methyldiazohydroxide which would be formed by methyl hydroxylationor carbon 4 hydroxylation, respectively, we compared the mutagenicities,without activation, of the corresponding model compounds, 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanoneand carbethoxynitrosaminomethane (methylnitrosourethane). Bothcompounds were highly mutagenic toward S. typhimurium TA 1535and TA 100, but at doses of 4 x 10^–3 to 4 x 10^–4µmol/plate,only 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone wasmutagenic. These results are consistent with those obtainedwith the deuterium substituted compounds and indicate the importanceof 4-oxo-4-(3-pyridyl)butylation of DNA in NNK mutagenesis.     

Phase I/II Trial of Nanomolecular Liposomal Annamycin in Adult Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia

BackgroundTreatment options for relapsed/refractory ALL in adult patients remain challenging. Annamycin is a highly lipophilic form of the anthracycline doxorubicin with the ability to bypass multidrug resistance mechanisms of cellular drug resistance.Patients and MethodsWe performed a phase I/II multicenter, open-label, study to determine the maximally tolerated dose (MTD) of nanomolecular liposomal annamycin in adult patients with refractory ALL.ResultsThirty-one patients were enrolled; the MTD was determined to be 150 mg/m²/d for 3 days. Other than tumor lysis syndrome, there were 3 grade 3 mucositis which comprised the MTD determination. There was also 1 case each of grade 3 diarrhea, typhlitis, and nausea. After determining the MTD, a 10-patient phase IIA trial was conducted. Eight of the patients completed 1 cycle of the 3 days of treatment at the MTD. Of these, 5 (62%) had an efficacy signal with complete clearing of circulating peripheral blasts. Three of these subjects also cleared bone marrow blasts with 1 subsequently proceeding onto successful stem cell transplantation.ConclusionSingle-agent nanomolecular liposomal annamycin appears to be well tolerated, and shows evidence of clinical activity as a single agent in refractory adult ALL.     

A Phase I Study of Fludarabine,Cytarabine, and Oxaliplatin Therapy in Patients With Relapsed or Refractory Acute Myeloid Leukemia

PurposeThe combination of cytarabine and fludarabine was associated with superior clinical outcomes compared with those of high-dose cytarabine in relapse acute myeloid leukemia (AML). We conducted a phase I study combining oxaliplatin with cytarabine and fludarabine therapy for patients with relapsed or refractory AML.Patients and MethodsBetween January 2008 and November 2009, 27 patients were registered in the study. Patients had histologically confirmed disease, performance status 0 to 2, and adequate organ function. The treatment regimen consisted of increasing doses of oxaliplatin (25, 30, or 35 mg/m²/d) on days 1 to 4 (escalation phase), and fludarabine (30 mg/m²) and cytarabine (500 mg/m²) on days 2 to 6, every 28 days for ≤ 6 cycles. The dose-limiting toxicity was defined as any symptomatic grade ≥ 3 nonhematologic toxicity lasting ≥ 3 days and involving a major organ system.ResultsOf 27 patients, 12 were treated in the dose-escalation phase and 15 at the maximum tolerated dose for oxaliplatin (30 mg/m²; expansion phase). All patients were evaluable for toxicity and response. Only 1 patient received the second cycle; the remaining patients received no further study treatment, owing to slow recovery from toxicities or physician decision. Grade 3-4 drug-related toxicities included diarrhea (grade 4) and elevated levels of bilirubin (grade 3) and aspartate transaminase (grade 3). In all, 3 patients had a complete remission and 2 patients complete response without platelet recovery.ConclusionOxaliplatin, cytarabine, and fludarabine therapy had antileukemic activity in patients with poor-risk AML, but it was associated with toxicity. Different schedules and doses may be better tolerated.     

A Phase I Trial of Chlorambucil Administered in Short Pulses in Patients with Advanced Malignancies

We carried out a phase I trial with chlorambucil. Thirty patients with advanced cancer were entered in six dose levels: 36, 48, 60, 84, 108, and 144 mg/m². The drug was given in six divided oral doses every 6 hours and the regimen was repeated every 3 weeks. The median age was 62 years (31-84), median Kamofsky performance status (KPS) 60 (40-90). All patients but one had received prior radiation therapy, chemotherapy, or both. Central nervous system toxicity was dose limiting, occurring in 5 of 6 patients at 144 mg/m². It was characterized by transient seizures, hallucinations, lethargy, stupor, and coma. Metoclopramide was successful in controlling nausea and vomiting, which was severe if the antiemetic was not used. Leukopenia (3 patients) and thrombocytopenia (2 patients) were mild. One patient with colorectal carcinoma had a minor response, and two patients with non-small cell lung cancer had stable disease. A safe dose for phase II trials is 108 mg/m² in six 6-hourly oral doses.     

Preliminary Phase II Study of 1-(2-Chloroethyl)-3-(MethylAlpha-D-Glucopyranos-6-Yl)-1-Nitrosourea (MCNU) Against Primary Lung Cancer and Metastatic Pulmonary Tumors

A phase II study of 1-(2-chloroethyl)-3-(methyl--D-glucopyranos-6-yl)-1-nitrosourea(MCNU) was conducted with 16 patients with primary lung canceror metastatic pulmonary tumors who had failed to respond toconventional therapy. MCNU was administered by a single intravenousinjection at a dose of 120 mg/m². There were no patients whoshowed any objective responses. Although stabilization was achievedin 12 patients, four patients with primary lung cancer experiencedprogressive disease. Gastrointestinal toxicities such as anorexia,nausea and vomiting were mild or moderate and readily subsidedwithout any treatment. The major toxic side effects were leukocytopeniaand thrombocytopenia. Five patients (38.4%) had leukocytopeniaof less than 2,000/mm³ and six patients (46.1%) had thrombocytopeniaof less than 5.0x10⁴/mm³.     

A Phase II Study of Midostaurin and 5-Azacitidine for Untreated Elderly and Unfit Patients With FLT3 Wild-type Acute Myelogenous Leukemia

BackgroundMidostaurin, a multikinase inhibitor, is approved for treatment of FLT3-mutant acute myeloid leukemia (AML). A phase I study established that midostaurin 75 mg orally twice daily for 14 days with standard dose azacitidine was safe and tolerable in elderly patients with AML. Herein, we report the phase II expansion cohort of previously untreated elderly or unfit patients with AML.Patients and MethodsPrimary objectives were to further describe the toxicity profile and determine the response rate in untreated patients with AML. Patients received midostaurin 75 mg orally twice daily on days 8 to 21 in combination with intravenous azacitidine at 75 mg/m² on days 1 to 7. Plasma inhibitory activity assay for FLT3 was performed pretreatment and on day 8 and day 15 of each cycle.ResultsTwenty-six patients (median age, 74 years; range, 59-85 years) with FLT3 wild-type AML were accrued. Patients received a median of 2 cycles of therapy (range, 1-10 cycles). Seven (29%) of 24 evaluable patients achieved a clinical response (4 complete response; 1 complete response with incomplete count recovery; and 2 partial response). The median overall survival was 244 days (95% confidence interval, 203-467 days). Hematologic, infectious, and gastrointestinal toxicities were comparable to similar studies. Peripheral blood FLT3 wild-type phosphorylation declined to 8% to 55% of pretreatment by day 15 of cycle 1 (7 patients) and declined with subsequent cycles (< 10% baseline) in 2 patients who were analyzed after cycle 3.ConclusionMultiple cycles of azacitidine and midostaurin were not well-tolerated, but persistent inhibition of FLT3 wild-type phosphorylation suggest intermittent dosing of midostaurin should be considered in future low-intensity regimens for FLT3-mutant AML.