The effects of the farnesyl transferase inhibitor FTI L-778,123 on normal, myelodysplastic, and myeloid leukemia bone marrow progenitor proliferation in vitro.

The effects of the farnesyl transferase inhibitor FTI-778,123 on the proliferation of normal, MDS, AML, and CML hemopoietic progenitor cells was studied. MDS myeloid and erythroid progenitors are significantly more sensitive to FTI than normal progenitors while AML myeloid progenitors may be somewhat more sensitive than normal progenitors. In contrast, no difference between CML and normal progenitors are detectable. These data strongly suggest that a trial of this agent in patients with MDS and perhaps in patients with AML is indicated.

A statistical approach to estimate O3 uptake of ponderosa pine in a mediterranean climate.

In highly polluted sites, stomatal behavior is sluggish with respect to light, vapor pressure deficit, and internal CO2 concentration (Ci) and poorly described by existing models. Statistical models were developed to estimate stomatal conductance (gs) of 40-year-old ponderosa pine at three sites differing in pollutant exposure for the purpose of calculating O3 uptake. Gs was estimated using julian day, hour of day, pre-dawn xylem potential and photosynthetic photon flux density (PPFD). The median difference between estimated and observed field gs did not exceed 10 mmol H2O m(-2) s(-1), and estimated gs within 95% confidence intervals. 03 uptake was calculated from hourly estimated gs, hourly O3 concentration, and a constant to correct for the difference in diffusivity between water vapor and 03. The simulation model TREGRO was also used to calculate the cumulative 03 uptake at all three sites. 03 uptake estimated by the statistical model was higher than that simulated by TREGRO because gas exchange rates were proportionally higher. O3 exposure and uptake were significantly correlated (r2>0.92), because O3 exposure and gs were highly correlated in both statistical and simulation models.

Abnormalities of molecular regulators of proliferation and apoptosis in carcinoma of the oral cavity and oropharynx.

OBJECTIVE: Abnormalities in genes regulating cell proliferation and death may affect disease outcome in squamous cell carcinoma (SCC) of the head and neck. METHODS: Proliferative activity (Histone H3 in-situ-hybridization (HISH) labeling index (LI)) and the genes and/or gene products of Cyclin D-1, c-erbB-2, Bcl-2, p21, and p53, were investigated in 35 patients with SCC of the oral cavity and oropharynx, previously studied for p27 expression. RESULTS: Overexpression or very low expression of Cyclin D-1 was associated with unfavorable disease outcome and shorter time-to-recurrence. High c-erbB-2 expression was significantly associated with shorter overall survival and was synergistic with low p27 expression. Bcl-2, HISH LI, p21 expression, and p53 mutation and protein analysis were not significantly predictive, but there were trends suggesting shorter disease-free/overall survival for patients with undetectable Bcl-2, high HISH, and mutant p53. CONCLUSIONS: Several cell proliferation and death regulators appeared to predict disease outcome. Limited evidence of cooperativeness among regulators was also seen.

O3 uptake and drought stress effects on carbon acquisition of ponderosa pine in natural stands.

• The effect of O3 exposure or uptake on carbon acquisition (net assimilation (A) or gross photosynthesis (Pg )), with and without drought stress, is reported here in 40-yr-old-ponderosa pine (Pinus ponderosa) trees. • Maximum daily gas exchange was measured monthly for 12 trees at four sites differing in pollutant exposure over two growing seasons with above- and below-average annual precipitation. Gas exchange measures were estimated between sampling periods using a generalized additive regression model. • Both A and Pg generally declined with cumulative O3 exposure or uptake at all sites. As a response variable, Pg was slightly more sensitive than A to cumulative O3 exposure. As a metric, O3 uptake vs exposure permitted slightly better statistical resolution of seasonal response between sites. • The effect of late summer drought stress was statistically significant only at the moderate pollution site, and combined synergistically with O3 exposure or uptake to reduce Pg . The general additive model allows the user to define a deleterious level of cumulative O3 exposure or uptake, and to quantitatively assess biological response.

P15INK4B gene methylation and expression in normal, myelodysplastic, and acute myelogenous leukemia cells and in the marrow cells of cured lymphoma patients.

P15INK4B methylation and expression was studied in bone marrow cells obtained from normal individuals, from patients who had been cured of lymphoma, and from patients with either MDS or AML. The level of p15 methylation was very low in normal BM cells and in CD34+ and CD34- subpopulations (0-6.5%; med, = 2.5%). P15INK4B transcripts were present in each of these cell populations. In contrast, methylation was the usual situation in MDS and AML marrows. The presence of methylation of the p15INK4B gene did not always indicate an absence of expression nor was expression always present if methylation was absent. P15INK4B methylation was studied in the marrows of nine patients (one studied twice) who had been cured of lymphoma and in whom hemopoiesis was believed to be normal. Increased methylaton was present in all 10 marrows. These data indicate that p15INK4B methylation is likely to be a very early event in the development of the secondary hematologic disorders.

Alterations in IRF1/IRF2 expression in acute myelogenous leukemia.

The interferon response genes 1 and 2 have been shown to be involved in the regulation of differentiation and proliferation of cells of the myeloid series, with the former functioning as an anti-oncogene and the latter as an oncogene. In the study described here, the levels of expression of these two genes and the ratio of their expression were compared in AML and normal marrow. The ratio of gene expression was significantly less in AML marrow cells as compared to normal marrow cells [med ratio = 1.33 vs. 2.97, P = 0.003]. While the expression ratio was unaffected by the presence or absence of either ras or fms mutations, p53 mutations were associated with higher IRF1:IRF2 expression ratios that wt p53 genes [med = 1.701 vs. 1.135, P = 0.014]. Given the functional characteristics and the competitive nature of these two genes, it is possible that leukemic transformation is associated with a fall in IRF1:IRF2 ratios. Finally, the administration of IL4 can result in the normalization of the IRF1:IRF2 ratio in the marrow cells of some patients with AML.

Poor prognosis acute myelogenous leukemia: 3--biological and molecular biological changes during remission induction therapy.

This is the third paper in a series which describes a new remission induction regimen for patients with 'poor prognosis' acute myelogenous leukemia (AML). Twenty-four patients were treated with two one day pulses of chemotherapy separated by 96 h. Each pulse consisted of two doses of cytarabine and a single dose of mitoxantrone. Amifostine was administered three times a week after the second pulse of chemotherapy until treatment outcome became known. The first paper described the outcome of treatment while the second described the relationship of treatment outcome to the pretherapy characteristics of the leukemia. This paper describes the changes in the leukemia cells which occur during remission induction therapy. While only a limited number of specimens were available for each post treatment study, the studies demonstrated a profound fall in blood counts, BM cellularity, and telomerase activity in leukemia cells after pulse #1 of treatment. This fall was usually accompanied by a coordinate rise in IL6, TNFalpha, and IL1beta transcripts within the AML cells which survived chemotherapy. High levels of telomerase activity in the day 5 marrow was correlated with high levels of IL1beta transcripts which in turn were associated with treatment failure ascribable to resistant disease.

Preparation of autologous leukemia and lymphoma vaccines expressing alpha-gal epitopes.

This study describes a novel method for increasing the immunogenicity of autologous tumor vaccines in leukemia and lymphoma patients by exploiting the natural anti-Gal antibody for in situ targeting of the vaccinating cells to antigen-presenting cells (APCs). Incubation of leukemia or lymphoma cells with neuraminidase and recombinant alpha 1,3-galactosyltransferase results in the synthesis of many alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on their cell membranes. Vaccination with such processed tumor cells results in the binding of the natural anti-Gal immunoglobulin G (IgG) antibody to these epitopes and opsonization of these cells for effective phagocytosis by APCs, such as dendritic cells and macrophages. These APCs may transport the vaccine to adjacent draining lymph nodes for subsequent effective processing and presentation of tumor-associated antigens (TAA) peptides to activate TAA-specific helper and cytotoxic T cells. Once the TAA-specific cytotoxic T cells are activated, they can leave the lymph node, circulate in the body, and seek metastatic cells expressing TAA to destroy them. Alternatively, activated helper T cells may provide the help required for B cells to produce antibodies to TAA on the leukemia or lymphoma cells. Because every patient receives his or her own TAA within the vaccinating cells, such vaccines are customized for the patient. These autologous tumor vaccines may be used as an adjuvant treatment that complements currently used treatment regimens by providing the immune system with an additional opportunity to be exposed effectively to autologous TAA.

Effectiveness of interferon-alfa and mid-cycle chemotherapy added to an anthracycline-based regimen in the treatment of aggressive non-Hodgkin's lymphoma.

Interferon-alfa in combination with cytotoxic chemotherapy has been shown to be effective in treating certain types of non-Hodgkin's lymphoma (NHL) (1). However, there is no published data on upfront induction treatment of aggressive NHL with IFN-alfa containing regimens. Studies have also shown that one can overcome regrowth resistance by administering mid-cycle agents which slow tumor proliferation between courses of cytotoxic therapy (2). Based on this, we treated 32 consecutive patients between 1/93 and 9/96 with a regimen containing cyclophosphamide 750 mg/m2, mitoxantrone 12 mg/m2, and teniposide 60 mg/m2 IV on day 1 with prednisone 100 mg PO given on days 1-5. On day 15, patients received vincristine 1.4 mg/m2 (2 mg max.) and bleomycin 10 units/m2 IV. Interferon-alfa-2b 5x10(6) units/m2 SQ was administered on days 22-26. The median age was 55 (range 26-83), M:F ratio was 2.5:1, and the median International Prognostic Index was 2. 38% of patients had stages I-II and 62% had stages III-IV disease. Fifty-nine percent of the patients achieved a complete response, 22% a partial response, and 19% had progressive disease. The overall survival (OS) was 81% and the progression free survival (PFS) was 56% at 4.3 years. There were no severe (grade IV) hematologic, flu-like, GI and infectious toxicities from IFN-alpha. Leukopenia was the main severe toxicity related to the chemotherapy regimen (days 1-15), but not IFN-alpha. Severe infection secondary to the chemotherapy regimen occurred in one patient. Interferon-alfa-2b and mid-cycle chemotherapy added to an anthracycline based regimen is effective induction treatment for patients with aggressive NHL. The OS and PFS using this regimen, based on regrowth resistance, appears to be at least as or more effective than CHOP therapy for this group of patients. Severe toxicities were rare.

Quantitation of interferon regulatory factor transcripts in patients with acute myeloid leukemia.

Interferon regulatory factors IRF-1 and IRF-2, the two mutually antagonistic factors, fluctuate during the cell cycle and play an important role in normal and neoplastic growth processes. The relative levels of these two transcripts were analyzed in 5 normal and 43 acute myeloid leukemia (AML) bone marrow (BM) specimens by a semiquantitative RT-PCR method. IRF-1 and IRF-2 cDNA sequences were coamplified using primers that were designed to span regions of high homology between the genes. Each primer can anneal equally to both IRF-1 and IRF-2 sequences. Hence, the relative amount of amplified products from each cDNA species provides an estimation of proportional concentration of the RNA transcripts in the test sample. Results indicate expression of both the transcripts on all the leukemia and lymphoma cell lines tested, normal and AML BM. Significantly higher IRF-1:IRF-2 ratio was observed in normal as compared to AML BM (p = 0.007). There was no correlation with clinical factors such as FAB subtype. A single dose of amifostine or three daily doses of recombinant IL-4 were administered to 5 and 8 AML patients, respectively. The changes in the expression of these transcripts were studied prior to administration of the agent (d0) and after 3 days (d3). IL-4 treatment showed significant increase in the IRF-1:IRF-2 ratio in 4 of 8 patients (p = 0.05); amifostine treatment did not show any appreciable change.

In vivo effects of IL-4, IL-10, and amifostine on cytokine production in patients with acute myelogenous leukemia.

Both IL-4 and IL-10 have been shown in vitro to inhibit leukemia cell secretion of IL-1beta, GM-CSF, and TNFalpha, and increase leukemia cell release of IL-1ra. In this study, we have investigated the in vivo effects of IL-4, IL-10, and amifostine on cytokine production in patients with acute myelogenous leukemia (AML). Serum IL-1ra, IL-1beta, TNFalpha, GM-CSF, and SCF levels were measured in AML patients who received IL-4, IL-10, or amifostine. No significant changes in the serum levels of IL-1ra, IL-1beta, TNFalpha, GM-CSF, and SCF were found in AML patients who received amifostine. Both IL-4 and IL-10 were found to increase serum IL-1ra. This data is in accord with the in vitro studies. However, IL-4 increased serum GM-CSF levels and IL-10 increased serum IL-1beta and TNFalpha levels. These in vivo effects of the two cytokines differ from their in vitro effects. Despite the similar effects of IL-4 and IL-10 on cytokine production by AML cells in vitro, different effects were observed in AML patients in vivo. IL-4 increased serum SCF levels, whereas IL-10 decreased serum SCF levels. IL-4 increased serum GM-CSF levels, whereas IL-10 had no effect on them. Although IL-10 increased serum IL-1beta and TNFalpha levels, IL-4 had no effect on them. These findings indicate that the in vitro effects of IL-4 and IL-10 do not necessarily reflect their in vivo effects, and that the complex effects of the two cytokines on serum cytokine levels make it difficult to predict their therapeutic potential.

High remission rate in acute myeloblastic leukemia with only two days of chemotherapy.

Twenty five patients with AML who had neither a history of toxic exposure or myelodysplasia were treated with a remission induction regimen consisting of two pulses of chemotherapy separated by 96 hrs. Each pulse consisted of cytarabine 2gm/m(2) (at t=0 and t=12 hrs) with mitoxantrone [30mg/m(2) ] administered immediately after the second cytarabine administration. Amifostine was administered three times a week [on Monday, Wednesday, and Friday] until the outcome of therapy was known. This regimen induced complete remissions in 15 of 17 patients less than 70 years of age and in 5 of 8 patients older than 70 years.

Effect of interferon-alpha on CD20 antigen expression of B-cell chronic lymphocytic leukemia.

Chimeric CD20 monoclonal antibody as alternative therapy in relapsed low-grade non-Hodgkin's lymphoma (NHL) has produced responses in nearly 50% of patients. Augmenting CD20 expression on tumor cells and/or inducing its expression may increase the cell kill and effectiveness of antibody therapy. Peripheral blood lymphocytes from 19 patients with B-cell chronic lymphocytic leukemia (B-CLL) were incubated in vitro in the presence of interferon-alpha (IFN-alpha) (500 U/ml and 1,000 U/ml) for 24 and 72 hours. The effect on CD20 expression was studied by flow cytometry. The differences in the percentage positivity, the mean fluorescence intensity (MFI), and the product of percentage positivity and MFI were used to assess upregulation. There was a significant upregulation of CD20 expression on B cells seen at both concentrations after 24-hour priming (p < 0.01). B-CLL cells cultured for 72 hours in the presence of IFN-alpha also showed upregulation of CD20 expression; however, the degree of upregulation was much lower than that seen at 24 hours. There was no statistically significant increase in CD20 antigen expression on normal lymphocytes following cytokine exposure. These results suggest that IFN-alpha priming may augment the effectiveness of antibody therapy by directly upregulating CD20 antigen expression in addition to its indirect action through effector cells of the host.

Poor prognosis acute myelogenous leukemia 2--biological and molecular biological characteristics and treatment outcome.

Biological and molecular biological studies were performed on the marrow cells of 25 patients with poor prognosis AML to both characterize this type of leukemia and to assess the relationship between the parameters which were measured and treatment outcome. Treatment failure associated with high levels of telomerase activity and low levels of IL6 transcripts. Studies of the effects of amifostine on these parameters demonstrated that this agent reduced telomerase activity in aspirates of AML marrows. These data suggest that the beneficial effect associated with the administration of amifostine after the end of chemotherapy is likely, to be due to a reduction in the rate at which the surviving leukemia cells repopulate the marrow.

Poor prognosis acute myelogenous leukemia: 1 - response to treatment with high dose cytarabine/mitoxantrone/ethyol @ (Amifostine).

Twenty patients with poor prognosis AML and four patients in the blastic phase of a myeloproliferative disorder were treated with two 'pulses' of therapy each consisting of two doses of high dose araC (separated by 12 h) followed by a single dose of mitoxantrone. The pulses were separated by 96 h. Amifostine was then administered tiw. The median age of the population was 68 years with 88% of patients having had either a prior MDS, MPD or toxic exposure. The acute leukemia of 58% of patients either entered a CR or reverted to preleukemic state. For patients under 70 years of age, treatment produced 62% CRs with a leukemia free decision marrow in 77%. For patients over 70 years the CR rate was 27% with 36% of patients having a leukemia free decision marrow.

Tumor necrosis factor modulates CD 20 expression on cells from chronic lymphocytic leukemia: a new role for TNF alpha?

Tumor necrosis factor alpha (TNF alpha) is a pleiotropic cytokine that is constitutively produced by leukemic cells in B Chronic Lymphocytic Leukemia (B-CLL). It has been shown to have autocrine and paracrine functions in normal B cells and in B lymphoproliferative diseases. This study was conducted to determine the effect of TNF alpha (in vitro) on CD20 expression on cells from patients with B-CLL. Currently, anti-CD20 monoclonal antibody therapy is becoming a second line treatment in the management of B cell disorders like low-grade non-Hodgkin's lymphoma (NHL) and B-CLL. Our results demonstrate amply that very low doses of TNF alpha (0. 0125 ng/ml) can be used to significantly increase CD20 expression on cells from patients of B-CLL as evidenced by increases in both percentage positivity and mean fluorescence intensity. The upregulation is evident as early as 24 hours and is maintained for up to 72 hours. We propose that the upregulation is a direct result of in vitro differentiation stimulated by TNF alpha. The results presented can be exploited in the designing of priming protocols prior to antibody therapy and this is discussed.

Cytokine production by in vitro processed and unprocessed haematopoietic cells.

The studies described here demonstrate that in vitro processing of cells before extraction of RNA has a major effect on the number and type of cytokine transcripts present within MDS and leukemia cells. Transcripts for GM-CSF, a cytokine whose production by leukemia cells is believed to play an important role in the pathogenesis of leukemia, was not detectable in 12/13 unprocessed AML specimens, in 12/12 MDS specimens, or in 7/7 CML specimens but once detected in many specimens after processing. These data strongly suggest that leukemia cell production of GMCSF rarely occurs in vivo.

Signal antonymy unique to myelodysplastic marrows correlates with altered expression of E2F1.

Myelodysplastic syndromes (MDS) have previously been reported to show competitively high rates of apoptosis and proliferation in the bone marrow (BM). Using a double-labelling technique in the present study, we demonstrated that a significantly high number of S-phase cells were simultaneously apoptotic (signal antonymy; SA) in MDS (mean +/- s.e.m. 53.5 +/- 6.7%, n = 24, P < 0.001). In contrast, SA was negligible in all other specimens studied, including normal control BM (n = 13) from non-Hodgkin's lymphoma (NHL) patients, BM from patients with de novo acute myelogenous leukaemia (1'AML; n = 5), or secondary AML that had transformed from MDS (2'AML; n = 10), or the solid tumours from patients with NHL (n = 9) or head and neck squamous cell carcinoma (HNSCC; n = 10). Subsequently, the expression of a transcription factor, E2F1, was studied in density-separated BM aspirate mononuclear cells from MDS patients (n = 9) and a normal control. Two separate sets of primers were used that recognized the regulatory retinoblastoma (Rb) protein-binding region and the functional DNA-binding region of E2F1. Interestingly, although the latter manifested the expected band (280 bp) in all samples, the Rb-specific primers showed the expected band (380 bp) in the normal and in 4/9 MDS specimens. Two other MDS specimens also showed a smaller band ( approximately 325 bp), whereas 3/9 MDS patients showed exclusively the smaller band. The levels of SA were significantly higher in those MDS cases that showed the smaller Rb-specific band either alone or in addition to the expected band (median 19.5%, n = 4, P = 0.037) than in those showing exclusively the expected band (median 0.4%, n = 3). Our present studies show SA as a characteristic feature of MDS and, importantly, demonstrate its link with an altered expression of E2F1 in some MDS patients.