Yasuyuki Kunieda

In vivo antitumor activity of herbimycin A, a tyrosine kinase inhibitor, targeted against BCR/ABL oncoprotein in mice bearing BCR/ABL-transfected cells

Herbimycin A, a benzoquinoid ansamycin antibiotic, has been shown to reverse the oncogenic phenotype of p60v-src transformed cells because of the inhibition of src protein tyrosine kinase. We previously demonstrated that herbimycin A displayed antitumor activity on the in vitro growth of Philadelphia chromosome-positive leukemia cells and BCR/ABL-transfected murine hematopoietic FDC-P2 cells through the inhibition of BCR/ABL protein tyrosine kinase. In this study, the transformed FDC-P2 cells were demonstrated to be tumorigenic in syngeneic DBA/2 mice. The intraperitoneal (i.p.) injection of the transformed tumor cells into DBA/2 mice induced infiltrations of abdominal organs, and then all of the mice died within time periods proportional to the cell numbers of inoculation. In mice that received an i.p. inoculation with greater than 1 x 10(5) cells, in vivo administration of herbimycin A by i.p. injection inhibited tumor formation and significantly prolonged survival time, and further, in mice inoculated with 1 x 10(4) cells, herbimycin A completely suppressed the in vivo growth of transformant FDC-P2 cells and brought about a complete remission. The present study revealed the in vivo efficacy of herbimycin A in mice bearing BCR/ABL-transfected cells.

Chronic myeloid leukemia presenting ALL-type BCR/ABL transcript

SummaryWe investigated the breakpoints of thebcr gene in 46 Ph1-positive CML cases by Southern blot analysis ofbcr rearrangement, and in 17 CML cases by a combination of Southern blot analysis and RT-PCR. By Southern blot, the breakpoint was not identified on M-bcr in three CML cases, of which one case showed the P210-typebcr/abl transcript and two cases showed the ALL-type (P190-type)bcr/abl transcript with or without P210 transcript. Later two cases showed unique hematological profiles such as thrombocytosis, mild myelofibrosis, and relative resistance to alkylating agents. Therefore, the present study suggests that expression of the P190-type transcript may affect clinical and hematological findings in CML.

Megakaryocytic differentiation of a leukemic cell line, MC3, by phorbol ester: Induction of glycoprotein IIb/IIIa and effects on expression of IL-6, IL-6 receptor, mpl and GATA genes

We investigated megakaryocytic differentiation in a newly-established Ph1-positive leukemic cell line, MC3, which showed tri-lineage immunophenotypes (myeloid antigens2+, CD19(1+) and CD41a1+) and was positive for CD34 and CD38. TPA induced MC3 cells to differentiate to an early stage of megakaryocyte lineage exhibiting an increase in the expression of platelet glycoproteins (GP) IIb/IIIa (CD41a), and an increase in cell size and nuclear ploidy. TPA treatment also enhanced the expression of GPIIb mRNA, and induced the expression of interleukin-6 (IL-6) and its receptor mRNAs, while it did not induce transcripts of the genes IL-11 and mpl ligand, and further decreased the transcript of the mpl gene. Consistent with these findings, MC3 cells treated with TPA showed an increased expression of GATA-1, but not GATA-3 transcripts, whereas those without TPA treatment expressed only the GATA-2 transcript. These results provide an insight into the study for the regulatory mechanism of megakaryocytopoiesis and leukemic cell differentiation.

Phase 1/2 clinical study of irinotecan and oral S-1 (IRIS) in patients with advanced gastric cancer

BackgroundIrinotecan and S-1, an oral fluoropyrimidine composed of tegafur, gimeracil, and oteracil potassium, have demonstrated antitumor activity against advanced gastric cancer. We performed a phase 1/2 study to determine the recommended dose, antitumor activity, and safety of a combination of S-1 and irinotecan in patients with advanced gastric cancer.MethodsPatients with previously untreated advanced gastric cancer were enrolled. Patients received irinotecan intravenously on days 1 and 15 plus oral S-1 twice daily on days 1–14 of a 28-day cycle. In the phase 1 part, the dose of irinotecan was escalated from 100 mg/m2 to 125 mg/m2 and then to 150 mg/m2.ResultsA total of 24 patients were enrolled. Overall, the median number of treatment cycles per patient was 5.9, and 92% of the patients completed at least two cycles. The overall response rate was 54.2% (13 of 24). The response rates in differentiated and undifferentiated cancer were 56.3% (nine of 16) and 50.0% (four of eight), respectively. Median survival time was 581 days. The maximum tolerated dose of irinotecan was not reached at the highest level. However, grade 4 neutropenia occurred at 125 mg/m2. We concluded that the recommended dose of irinotecan for the present regimen was 125 mg/m2.ConclusionTreatment with S-1+irinotecan is considered effective in patients with advanced gastric cancer who have not previously received chemotherapy. A combination of irinotecan and S-1 was well tolerated in patients with advanced gastric cancer and could be given on an outpatient basis.

Analysis of the p53 gene mutations in acute myelogenous leukemia : the p53 gene mutations associated with a deletion of chromosome 17

In order to determine the relevance of the p53 tumor suppressor gene mutations in acute myelogenous leukemia (AML), we analyzed the p53 gene in genomic DNA of 18 unselected cases of AML by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. We detected three cases (16.7%) with the p53 gene mutations showing only the mutant alleles; the high incidence in cases with loss of a whole chromosome 17 (two of three) contrasted with the low incidence in cases without abnormalities of chromosome 17 (one of 15). These cases containing the mutations of the p53 gene showed a poor prognosis. Although we analyzed a rather small series of patients, these findings suggest that the p53 gene mutations might be involved in the progression and prognosis of at least some cases of AML.

Establishment and Characterization of a New Ph1-Positive Chronic Myeloid Leukemia Cell Line MC3 with Trilineage Phenotype and an Altered p53 Gene

A new Ph1-positive leukemic cell line (MC3) expressing the P210bcr/abl oncoprotein was established from a patient with CML in blast crisis. The MC3 cells showed the trilineage phenotype of myeloid, lymphoid (CD19) and megakaryocytoid lineages, and had a proliferative response to rhIL-1 and rhIL-3 in the serum-free culture. These results and the expression of CD34 indicated that the MC3 cells have characteristics of hematopoietic progenitor cells. Recently, it has been documented that alterations of the p53 gene in leukemic cells are frequently detected during the blast crisis of CML. The MC3 cells contained the altered p53 gene. In addition, the original leukemic cells showed the point-mutational activation of the N-ras gene and an additional chromosomal abnormality inv(3q), but the MC3 cells contained no such abnormalities, indicating that not all of the original leukemic cells had these abnormalities. Thus, the MC3 cell line may provide several insights into investigations of the blast crisis in CML as well as hematopoietic progenitor cells.

Effects of rhG-CSF on Infection Complications and Impaired Function of Neutrophils Secondary to Chemotherapy for Non-Hodgkin's Lymphoma

It has been previously demonstrated that the administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF) ameliorates the decrease of the polymorphonuclear neutrophils (PMNs) count after the cytotoxic chemotherapies, thereby reducing the infection complications associated with neutropenia. In this multi-center study, we studied the prophylaxtic effect of rhG-CSF administration on infection complications in patients with non-Hodgkin malignant lymphoma, who received cytotoxic chemotherapies (CHOP or ProMACE/CytaBOM). rhG-CSF administration reduced the frequency of infection complications, and there was no obvious difference in its frequency between the CHOP-treated and the ProMACE/CytaBOM-treated groups when administered with rhG-CSF, thereby indicating that third generation therapy for NHL may be safely completed in Japanese in combination with rhG-CSF administration. Furthermore, we investigated both the in vitro and the in vivo effects of rhG-CSF on the function of PMNs in patients with NHL and healthy donors, and revealed that the administration of rhG-CSF for NHL patients receiving cytotoxic chemotherapy brought on an improvement of the production of active oxygen but did not affect serum levels of IFNs, IL-1-beta, and IL-6, inspite of a slight elevation of TNF-alpha. Consistent with these results, in vitro treatment of PMNs with rhG-CSF induced no significant production of these inflammatory cytokines and their mRNA expressions. Furthermore, rhG-CSF administration showed no significant effects in vivo on the expression of CD11a, CD11b and LECAM-1 on PMNs and integrins on platelets.

Ultra-high level of serum soluble interleukin-2 receptor at diagnosis predicts poor outcome for angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of non-Hodgkin lymphoma and displays an aggressive clinical course with poor outcome. To identify prognostic factors for AITL, we retrospectively analyzed 36 patients with AITL. The median age was 74 years with 83% of the patients having advanced stage. Eighty-three percent received CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone)-like chemotherapies, resulting in an overall response rate of 63%. With a median follow-up of 9 years, the estimated overall survival at 5 years was 33.3%. Median serum level of soluble interleukin-2 receptor (sIL-2R) was 5615 U/mL at diagnosis, and over 10 000 U/mL of sIL-2R was identified as a significant poor prognostic factor, independent of the International Prognostic Index, Prognostic Index for peripheral T-cell lymphoma and Prognostic index for AITL (hazard ratio [HR], 4.42; 95% confidence interval [CI], 1.49–13.11; log-rank, p < 0.01). Our study shows that an ultra-high level of serum sIL-2R at diagnosis is a significant poor prognostic biomarker for AITL.

Modified-irinotecan/fluorouracil/levoleucovorin therapy as ambulatory treatment for metastatic colorectal cancer: results of phase I and II studies.

AbstractBackground: Combined therapy with irinotecan/fluorouracil/levoleucovorin (calcium levofolinate) [IFL] has lost its position as the standard regimen for metastatic colorectal cancer because its toxicity and effectiveness have become controversial. Objective: To (i) identify the optimal regimen for IFL therapy in terms of irinotecan dosage, and (ii) determine the maximum tolerated dose and efficacy of the modified-IFL regimen in patients with histologically confirmed advanced colorectal cancer. Methods: In a phase I study, nine patients with advanced colorectal cancer received IFL treatment modified such that irinotecan was administered every 2 weeks, as opposed to the more toxic once-weekly administration. The study evaluated three escalating dose levels of irinotecan (100,125 and 150 mg/m2). Each treatment cycle consisted of irinotecan on days 1 and 15; fluorouracil 600 mg/m2 on days 1,8, 15 and 22; and levoleucovorin 250 mg/m2 on days 1, 8, 15 and 22. Data from the phase I study were used to determine the recommended dose of irinotecan for the phase II study. The latter study evaluated the effectiveness (overall response rate, median time to disease progression and median survival time) and tolerability of this modified-IFL therapy as ambulatory treatment in 22 patients with advanced colorectal cancer. Results: The dose-limiting toxicity of irinotecan was grade 3 neutropenia, which occurred in three patients at dose level 2 (125 mg/m2); furthermore, a fourth patient developed grade 4 neutropenia at this dose level. Therefore, 125 mg/m2 was considered to be the maximum tolerated dose, and the dose of irinotecan for the phase II study was set at 100 mg/m2. Fourteen patients achieved partial response using this modified-IFL regimen, and the overall response rate was 63.6% (95% CI 43.5, 83.7). The median time to progression was 197 days (range 111–283 days) and the median survival time was 414 days (95% CI 116, 712). Toxicities were acceptable and manageable. Conclusions: Modified-IFL therapy is a practical, effective and tolerable option for ambulatory treatment of advanced colorectal cancer.

Effect of granulocyte colony-stimulating factor on IL-12 p40 production during chemotherapy for B-cell lineage non-Hodgkin's lymphoma patients

Abstract:  Interleukin (IL)‐12 is a 70‐kDa cytokine comprised of two disulfide‐linked proteins (p35 and p40) and is essential for the initiation of effective immune response. Granulocyte‐colony stimulating factor (G‐CSF) affects the balance in the production of anti‐inflammatory cytokines. We investigated the serum IL‐12 p40 and IL‐12 Mix (p40 and p70) production in 28 patients with B‐cell lineage non‐Hodgkins lymphoma (NHL) treated with chemotherapy (e.g., CHOP regimen) with or without G‐CSF administration and eight healthy volunteers. We found that serum levels of IL‐12 p40 (191.2 ± 150.0 pg/mL) and IL‐12 Mix (277.4 ± 274.5 pg/mL) in the patients before chemotherapy were higher than those in the healthy volunteers (IL‐12 p40: 76.4 ± 25.3 pg/mL, IL‐12 Mix: 48.5 ± 33.4 pg/mL) (P = 0.04 and 0.02, respectively). Next, we examined the serum IL‐12 p40 and IL‐12 Mix levels in nine patients receiving chemotherapy with administration of G‐CSF (CG group, n = 9) and without G‐CSF (C group, n = 9). Serum IL‐12 p40 and IL‐12 Mix levels were decreased on 10 d after chemotherapy in both groups, and those in CG groups were significantly lower than those in C group. These results indicated that administration of G‐CSF decreased serum IL‐12 p40 and IL‐12 Mix levels. Overall survival (OS) at 24 months was not significantly different in the two groups (58.3% in group C vs. 80.0% in group CG, P = 0.67). However, the survival rate of patients at clinical stages III and IV in CG group (n = 6, 66.0%) was significantly better than that of patients in C group (n = 4, 25.0%) (P = 0.02). Long‐term administration of G‐CSF appears to influence the survival rate by reducing immunosuppressive IL‐12 p40 production.