Guido Tricot

The role of aggressive chemotherapy in the treatment of the myelodysplastic syndromes

Fifteen patients with the initial diagnosis of myelodysplastic syndrome (MDS) received aggressive chemotherapy with high dose cytarabine or with a standard acute myeloid leukaemia (AML) regime. Cases treated with aggressive chemotherapy were either younger individuals with refractory anaemia with excess of blasts (RAEB) or patients, irrespective of age in advanced stages of MDS (RAEB in transformation or after evolution to frank AML), who did not have a major infection at the time of presentation. Age seemed to be the most important factor in determining the outcome of aggressive remission induction chemotherapy in MDS: 86% of the patients <50 years entered complete remission, compared to only 25% in the older age group. In spite of intensive consolidation therapy the duration of complete remission was short. We conclude that young patients (<50 years) with excess of bone marrow blasts should be treated with aggressive chemotherapy even in the early stages of the disease. Elderly patients in advanced stages of MDS should be treated with less aggressive chemotherapy.

EVOLUTION OF THE MYELODYSPLASTIC SYNDROMES

The myelodysplastic syndromes (MDS) are imprecisely defined haematological dsorders that may precede acute myeloid leukaemia (AML), sometimes by many years. MDS mainly affect older individuals and are characterized by one or more cytopenias in the peripheral blood, while the bone marrow is usually normoor hypercellular. In contrast to AML, where the leukaemic cells show very limited or no differentiation, but are rapidly expanding, in MDS the haematopoietic cells always display some degree of differentiation and the disease expands more slowly. The diagnosis of the early stages of MDS may be difficult and no generally accepted diagnostic criteria for MDS are available at present, leading to the risk that MDS may be considered a common denominator for all unexplained cytopenias and bone marrows, which are difficult to interpret. While survival of untreated AML patients is very short with a median duration of 2 months (Tivey, 1955), the natural evolution in MDS is largely unpredictable. Some patients will experience prolonged survival, whereas a substantial number of patients will die within the first year after diagnosis (Vallespi et al , 1985; Tricot et al, 19 8 5a). The question why evolution in MDS may be so different has not yet been resolved and the opinion still largely prevails that MDS is a premalignant condition, clearly different from AML.

KRAS2 oncogene overexpression in myelodysplastic syndrome with translocation 5;12

The factors that initiate and maintain the abnormal hematopoietic clone in the myelo-dysplastic syndromes (MDS) remain largely unknown. We describe a patient with MDS associated with an abnormal karyotype, 46,XY,t(5;12)(q31;p12). According to the FAB cooperative group classification, the patient was classified as chronic myelomonocytic leukemia. Because of the particular chromosomal translocation, the structure-function relationship of three genes relevant to the translocation breakpoints, CSF2, FMS, and KRAS2, was studied in bone marrow and peripheral blood lymphocytes in this patient. No major structural alterations were observed at these three genetic loci. Although the levels of expression of the CSF2 and FMS genes remained unaltered, the KRAS2 oncogene was overexpressed approximately six-fold in bone marrow cells from the MDS patient compared with normal donors. We postulate that the RAS oncogene activation may be instrumental in the genesis of MDS.

Enzyme-pattern-targeted chemotherapy with tiazofurin and allopurinol in human leukemia

The hypothesis was tested that the increased IMP dehydrogenase activity in human myelocytic leukemic cells, and along with it guanylate biosynthesis, might be a sensitive target to chemotherapy by tiazofurin. 1. IMP dehydrogenase activity in normal leukocytes was 3.1 +/- 0.5 (means +/- S.E.) nmol/hr/mg protein and in leukemic cells it was elevated 15- to 41-fold. The activity of guanine phosphoribosyltransferase in normal leukocytes was 389 +/- 27 nmol/hr/mg protein and in the leukemic cells it increased 2.8- to 6.8-fold. 2. IMP dehydrogenase was purified 4,900-fold to homogeneity from rat hepatoma 3924A with a yield of 30%. The kinetic properties of the hepatoma enzyme were similar to those of the enzyme in human myelocytic leukemic blast cells because of the similarity of the Kms for IMP (23 microM), NAD (44 and 65 microM); the Ki for TAD was 0.1 microM in both enzymes. 3. There was a selectivity of the in vitro response to tiazofurin in human normal and leukemic leukocytes. When labeled tiazofurin was incubated with leukocytes from normal, healthy volunteers and from leukemic patients, the leukemic leukocytes made 20- to 30-fold more TAD and the GTP content decreased as compared to normal leukocytes. This procedure proved to be a suitable predictive test in a clinical setting because patients with positive tests responded to tiazofurin whereas those with negative ones did not. 4. The National Cancer Institute approved a chemotherapeutic phase I/II trial which concentrates on treatment of refractory acute myelocytic leukemia. Tiazofurin is infused in a 60-minute period with a pump to insure uniform delivery. A novel aspect of the trial was that it was directed primarily by the biochemical impact of tiazofurin on IMP dehydrogenase activity and GTP concentration and the tiazofurin doses were to be adjusted accordingly. Patients received allopurinol as a routine precaution against possible accumulation of uric acid in the kidney. 5. In the first eight patients, there was one complete remission, two entered the chronic phase, two entered into partial remission, one did not respond, and two were not evaluable. In the five patients who responded, there was a rapid, profound decrease in IMP dehydrogenase activity of the blast cells and a gradual decline in GTP concentrations. The blast cell count followed the decrease in the GTP concentration. The white blood cell count was largely preserved. 6. Bone marrow aspirates and peripheral blood samples showed that with tiazofurin treatment there was an induced differentiation of the myelocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of de novo and salvage pathways in chemotherapy

An overview was presented of our approach of inhibition of de novo and salvage pathways in pyrimidine and purine metabolism. 1. Combination of acivicin, an inhibitor of de novo biosynthesis, and dipyridamole, a transport inhibitor, provided synergistic cytotoxicity in hepatoma and colon carcinoma cells. 2. AZT, a competitive inhibitor of the salvage enzyme, thymidine kinase, and 5-FU or MTX provided synergistic cytotoxicity in hepatoma 3924A. In human colon carcinoma HT-29 cells AZT and methotrexate yielded synergistic cytotoxicity and thymidine and hypoxanthine together provided protection from the action of these drugs. 3. These observations are significant because in rat hepatoma 3924A and in human cell lines HT-29, HL-60 and K562 thymidine kinase activity was 16- to 67-fold higher than that of dTMP synthase. Therefore, inhibition of dTMP synthase activity alone may provide poor responses because the salvage pathways can circumvent this block. 4. In leukemic patients treated with tiazofurin, an inhibitor of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, and with allopurinol, which inhibits GPRT activity through raising plasma hypoxanthine levels, synergistic therapeutic results were obtained. The responses in sensitive patients entailed a decrease in IMP dehydrogenase activity and GTP concentration in leukemic cells and down-regulation of the ras and myc oncogenes. The down-regulation of the ras oncogene by tiazofurin through the decrease of GTP concentration has now been shown in K562, HL-60 and hepatoma cells and in patients with chronic granulocytic leukemia in blast crisis. Tiazofurin may be useful in studies on selective depression of the expression of the ras oncogene. 5. In 27 consecutive patients 50% responded positively to tiazofurin treatment. From this group, 10 out of 12 patients (83%) with chronic granulocytic leukemia in blast crisis responded to tiazofurin treatment.

c-MYC and c-MYB expression in acute myelogenous leukemia

Monoclonal antibodies and flow cytometry were used to detect the expression of c-myc and c-myb in the bone marrow (BM) and peripheral blood (PB) cells of patients with acute myelogenous leukemia (AML). The expression of neither gene was correlated with the percent blast cells in the BM or PB nor was there a correlation between c-myc and c-myb expression. A wide range of expression of each gene was found within each FAB type of AML. Patients who had a high proportion of leukemia cells expressing c-myb were less likely to respond to remission induction therapy than patients in whom a low proportion of cells expressed c-myb. This association appears to reflect an inverse relationship between the proportion of cells expressing c-myb and the sensitivity of leukemia cells to the killing effects of chemotherapy in vivo. Treatment outcome was unrelated to c-myc expression.

Pregnancy-associated aplastic anemia--report of 3 cases.

Abstract:  3 patients with pregnancy‐associated aplastic anemia are reported. Management throughout most of the pregnancy consisted of supportive care. In 2 patients an improvement in blood counts occurred after delivery and, in 1 of these, the pancytopenia recurred during a subsequent pregnancy. In 1 case no improvement occurred after delivery and the patient ultimately required an allogeneic bone marrow transplantation. The outcome of these 3 cases demonstrates that pregnancy‐associated aplastic anemia can be managed successfully. The improvement that often occurs after delivery suggests a pathogenetic role for pregnancy in the development of this disease.

Prognostic factors in the myelodysplastic syndromes

The myelodysplastic syndromes are acquired clonal hematologic malignancies characterized by progressive cytopenia and an increased risk of evolution to acute myeloid leukemia. It mainly affects elderly people, but may also be found in younger patients and children. MDS represents a heterogeneous group of disorders. Some patients will experience prolonged survival, whereas a substantial number of patients will die within the first year after diagnosis. Treatment of patients should be based on life expectancy. Patients with pancytopenia, excess of bone marrow blasts, complex chromosome abnormalities, abnormal chromosome 7, older age and secondary MDS have a poor prognosis. Several simple scoring systems are available, based on peripheral counts, percent of bone marrow blasts and age, that provide significant prognostic information about life expectancy in patients with MDS. The most widely used is the Bournemouth scoring system. The prognostic factors and the scoring systems are reviewed.

IMP dehydrogenase: Inhibition by the anti-leukemic drug, tiazofurin

Tiazofurin through its active metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) inhibits IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis. IMP dehydrogenase activity in human leukemic cell extracts (33.4 +/- 0.1 nmol/h/mg protein) was increased 11-fold compared to normal leukocytes (3.1 +/- 0.5). Km values for IMP and NAD+ of leukemic IMP dehydrogenase were 22.7 and 44.0 microM, respectively. XMP inhibited competitively with IMP and noncompetitively with NAD+. NADH exerted mixed type inhibition with respect to both IMP and NAD+. The inhibitory pattern of TAD was quite similar to that of NADH; however, the affinity of TAD to leukemic IMP dehydrogenase (Ki = 0.1 microM) was three orders of magnitude higher than the natural product NADH (Ki = 150 microM). These results contribute to an understanding of the mechanism of action of tiazofurin in the treatment of leukemia.

Clinical and molecular impact of inhibition of IMP dehydrogenase activity by tiazofurin

The impact of tiazofurin on inhibition of IMP dehydrogenase was discussed at the clinical and molecular levels. 1. Evidence was provided for the role of IMP dehydrogenase and guanylates in the expression of the neoplastic program in cancer cells with particular relevance to human leukemic cells. 2. The argument for expecting an impact of tiazofurin in human myelocytic cells was provided. 3. Similarity of the kinetics of human leukemic cell IMP dehydrogenase to the rat hepatoma enzyme was documented. 4. New evidence was provided for the role of salvage in chemotherapy and the function of hypoxanthine in inhibiting guanine salvage. 5. The action of tiazofurin and retinoic acid was reported in HL-60 leukemic cells. 6. The effect of tiazofurin and retinoic acid on proliferation and cytotoxicity was outlined for hepatoma 3924A cells. 7. The effect of guanine on induced differentiation by tiazofurin and retinoic acid was examined. 8. Biochemical basis was provided for the lack of development of resistance in patients treated with tiazofurin. 9. Presumptive evidence was provided that tiazofurin treatment induced differentiation of leukemic cells in the patients. 10. The molecular biology of tiazofurin-induced differentiation in K-562 cells was reviewed with the possible relevance to clinical treatment that tiazofurin might also act through down-regulation of ras oncogene.