Oral vitamin C supplementation to patients with myeloid cancer on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes.

BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.

Anti-angiogenic potential of Gleditsia sinensis fruit extract.

Blood supply plays a crucial role in solid tumour development and leukaemogenesis. It has been suggested that blocking of angiogenesis could be possible in cancer therapy. We have demonstrated the antiproliferative activity of Gleditsia sinensis fruit extract (GSE) on various human solid tumour cancer cell lines as well as leukaemia cell lines and primary cultured leukaemia cells obtained from leukaemia patients. However, the antiangiogenic potential of GSE has not been demonstrated. Here we demonstrated that GSE could reduce vascular endothelial growth factor (VEGF) mRNA expression in dose- and time course-dependently in MDA-MB231 breast cancer and HepG2 hepatoblastoma cell lines as measured by reverse transcriptase polymerase chain reaction. Enzyme-linked immunosorbent assay further showed that GSE could reduce the VEGF secretion from various cancer cell lines including MDA-MB231, HepG2, HL-60 (acute promyelocytic leukaemia) and eleven primary cultured leukaemia cells obtained from acute myelogenous leukaemia patients. In vivo chick chorioallantoic membrane assay illustrated that GSE could reduce the angiogenic activity of basic fibroblast growth factor. Taken together, the information suggested that GSE could be potentially used as an angiogenic inhibitor in both solid tumour and leukaemia therapy.

Minimal toxicity to myeloid progenitor cells of weekly 24-hr infusion of high-dose 5-fluorouracil: direct evidence from colony forming unit-granulocyte and monocyte (CFU-GM) clonogenic assay.

Although very high doses of 5-fluorouracil was used in the weekly 24-h infusion, high-dose 5-fluorouracil (2600 mg/m2/week) and leucovorin (500 mg/m2/week) protocol, myelosuppression was surprisingly low. The current study was conducted to investigate the possible mechanism underlying the low myelosuppression. To mimic the clinical situation, peripheral blood progenitor cells collected from 12 patients were used for colony forming unit-granulocyte and monocyte clonogenic assay; and 2 representative modes of 5-fluorouracil exposure (30 min. versus 24 hr) were examined for cytotoxic effects on human myeloid progenitor cells. Previous pharmacokinetic studies have estimated the concentrations of 5-fluorouracil in the bone marrow to be 200-400 microM and 1-2 microM for the 30 min. infusion (600-900 mg/m2) and the 24 hr-infusion (1000-2000 mg/m2) regimens, respectively. The results of our colony-forming unit-granulocyte and monocyte clonogenic assay showed that 24-hr exposure to 5-fluorouracil (2 microM) and 30 min. exposure to 5-fluorouracil (100 microM) resulted in 27.2% and 78.2% inhibition of the colony formation, respectively. Our data provided direct evidence which may explain why myelotoxicity is significantly less in weekly 24 hr infusion of fluorouracil than in the conventional bolus regimens.

Competitive reverse transcriptase-polymerase chain reaction shows that dietary zinc supplementation in humans increases monocyte metallothionein mRNA levels.

Zinc status is difficult to evaluate in humans. Metallothionein gene expression is transcriptionally regulated by dietary zinc and thus could serve as an assessment parameter based on zinc-dependent function. We used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to establish that MT mRNA is increased in a human monocytic cell line by addition of zinc to the medium. To examine this response in human subjects, a dietary supplement of 50 mg zinc gluconate/d was given for 15 d. Monocytes were purified from venous blood using NycoPrep 1.068. Monocyte purity was determined by flow cytometry using fluorescent anti-human monocyte CD14 antibodies. Total monocyte RNA was extracted and converted to cDNA by reverse transcription. Competitive RT-PCR was used to analyze differences between cDNA levels that are proportional to MT mRNA levels in monocytes from zinc-supplemented and control subjects. RT-PCR oligonucleotide primers were designed to amplify both a 201 bp segment of the human MT cDNA and a 180 bp competitor cDNA template. The 180 bp competitor cDNA template was used for MT cDNA quantitation. The RT-PCR data show that there was a significant increase in monocyte MT mRNA in subjects within 6 d of zinc supplementation, which remained elevated at d 15 of supplementation. In contrast, plasma zinc was greater at d 6 of zinc supplementation, but by d 15 of supplementation, while still elevated, was close to control levels. These data suggest that monocyte MT mRNA levels respond to zinc supplementation and that the response could serve as a more useful assessment variable than plasma zinc for the measurement of zinc status in humans.

Adverse prognostic features in 251 children treated for acute myeloid leukemia.

Potential predictors of event-free survival (EFS) were assessed in 251 consecutively diagnosed children treated for acute myeloid leukemia (AML) on three successive clinical trials. The lack of significant differences in 4-year EFS for these studies (20% +/- 4%, 29% +/- 4%, and 20% +/- 7%) permitted combined analysis of presenting features. Splenomegaly (P = .002), coagulation abnormalities (P = .001), leukocyte count > or = 10 x 10(9)/L (P = .002), and age > 14 years (P = .01) were statistically significant predictors of a poorer EFS by univariate analysis and retained significance in multivariate analysis. Age < 2 years and monocytic leukemias (often cited as adverse factors in AML) showed no prognostic influence in this study. The estimated relative risk of failure for a child with a single adverse feature at diagnosis was at least 1.4 times greater than that for a patient with no adverse features. For children with two or more adverse features, the relative risk increased by more than threefold. These clinical variables, alone or in combination, may identify important subgroups of patients with AML at high risk for failure and for whom improved or alternative therapies are especially important.

[Therapeutic effectiveness of vitamin D3 in patients with myelodysplastic syndromes, leukemias and myeloproliferative disorders].

We tried to treat 13 patients with myelodysplastic syndromes (MDS), leukemias and myeloproliferative disorders, with alfacalcidol for their hematological improvement. Eight of them had MDS, 2 acute leukemia (M3, M4), 1 chronic myelogenous leukemia and 2 primary myelofibrosis. All patients were untreated except for 3 patients (PASA, RAEB, AML-M4) who had been treated with mepitiostane, prednisolone and BH.AC-AMP regimen, respectively, prior to alfacalcidol therapy. All patients received alfacalcidol orally for at least one month. The dosage of alfacalcidol ranged from 0.25 to 10 micrograms/day, and the medicine was administrated intermittently when the dosage exceeded 6 micrograms/day to prevent hypercalcemia. The therapeutic effectiveness of alfacalcidol was evaluated according to a criteria by Koeffler (Cancer Treat Rep 69: 1399, 1985) with minor modifications. Three patients (PASA, RAEB, CMML) showed partial response, 3 (RAEB, RAEB in T, AML-M4) minor response and rest of the patients did not respond. The hematological improvement of 6 responders was transient (from 1 to 2 months), however, one patient (PASA) is still responding to alfacalcidol therapy (0.25 microgram/day) for over 12 months. The dysplastic features of hemopoietic cells in the bone marrow showed no noticeable change during the hematological improvement in these responders, suggesting the improvement was obtained as a result of alteration in the proliferation or differentiation of neoplastic clone. None of 13 patients developed hypercalcemia. One patient (AML-M4) became excitable on high dose alfacalcidol (10 micrograms/day). In conclusion, alfacalcidol therapy is effective in some patients with MDS or leukemias and appears worthy especially in the clinical state in which chemotherapy is not indicated.

Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using percoll density gradients and elutriation.

Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU-GM and CFU-GEMM could be enriched 75-fold in a light density fraction of d less than 1.056 g/ml and the technique could be adapted to cope with more than 10(10) buffy coat leucocytes. Progenitors cells were concentrated 3-fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity and between 5% and 40% of these cells formed CFU-GM or BFU-E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI-3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level.

Marked hypophosphatemia associated with acute myelomonocytic leukemia. Indirect evidence of phosphorus uptake by leukemic cells.

A 51-year-man was seen with the diagnosis of acute myelomonocytic leukemia. The WBC count was 380,000/microL at presentation. The serum phosphorus concentration was 0.4 mg/dL and 0.2 mg/dL prior to any phosphorus replacement. Urinary phosphorus excretion was too low to be measured. The patient did not demonstrate any of the usual causes of profound hypophosphatemia with hypophosphaturia. The parathyroid glands were normal at necropsy. Reasons for believing the profound hypophosphatemia was due to phosphorus uptake by the leukemic cells are discussed.

Haematological reconstitution after autografting for chronic granulocytic leukaemia in transformation: the influence of previous splenectomy.

Peripheral blood values and bone marrow appearances were monitored in eight patients treated for chronic granulocytic leukaemia in transformation by cytotoxic drugs with or without total body irradiation followed by autografting with cryopreserved-thawed peripheral blood nucleated cells. One of the patients was 'autografted' on two occasions. Five patients had been splenectomized early in the first chronic phase and the other three patients had their spleens intact. Recovery of peripheral blood values was more rapid in the splenectomized than in the non-splenectomized patients. CFUc were present in the circulation immediately after autografting in each case but subsequently the pattern of CFUc changes differed between patients. The bone marrow was hypocellular at the time of autografting but the rate at which it returned to a typical chronic phase picture varied. Peripheral blood nucleated cells collected at the time of diagnosis include stem cells with the capacity to repopulate the marrow after 'ablative' therapy for transformation. Elective splenectomy in the chronic phase may promote more rapid recovery of peripheral blood values but its long-term importance is unknown.

Cryopreserved peripheral blood cells functioning as autografts in patients with chronic granulocytic leukaemia in transformation.

Six patients with chronic granulocytic leukaemia (CGL) in transformation were treated with cytotoxic drugs or cytotoxic drugs plus total body irradiation, followed by infusion of reconstituted autologous peripheral blood cells that had been collected from them at diagnosis and stored in liquid nitrogen for up to 58 months. In four cases the blood and bone-marrow appearances were rapidly restored to those of typical chronic-phase disease. In three of these patients transformation recurred at 74, 32, and 26 weeks respectively. One patient was still in second chronic phase at eight weeks. One of the patients who entered a second transformation was restored to a third chronic phase by further treatment with cytotoxic drugs and a second autograft. Cryopreserved autologous blood cells may thus restore some patients with CGL in transformation to chronic-phase disease and so may help to prolong life.

Cap formation on lymphocytes from patients with leukemic diseases induced by four different lectins.

When ficoll purified peripheral blood lymphocytes were treated with fluorescein conjugated lectins from lentils (LCH), castor beans (RCA) and phaseolus coccineus beans (L-and E-PHA) for 15 min and the percentages of the cap forming cells were examined, the values of leukemic lymphocytes were reduced compared to the values obtained with normal lymphocytes. The reduction was more than half in patients with acute and chronic myelogenous leukemia and immunoblastoma, it was only one quarter in patients with chronic lymphocytic leukemia, Hodgkin's disease and lymphosarcoma. The lowest number of cap forming cells was found in lymphoblasts of established lymphoblastoid cell lines. The four different lectins showed nearly the same capacity in the induction of caps. After successive binding, the different lectins showed cocapping on the lymphocyte surface.