Efficacy and safety of imatinib mesylate (Glivec) in combination with interferon-alpha (IFN-alpha) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL).

Imatinib has marked antileukemic activity in advanced Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), but secondary resistance develops rapidly, reflecting the limitations of single-agent therapy. Experimental data suggest that interferon-alpha (IFN-alpha) enhances the antileukemic activity of imatinib. We therefore examined combined imatinib and low-dose IFN-alpha in six patients with Ph+ALL who were ineligible for stem cell transplantation. All patients had received imatinib for 0.5-4.8 months prior to IFN-alpha, for relapsed (n=3) or refractory (n=1) Ph+ALL or as an alternative to chemotherapy following severe treatment-related toxicity (n=2). Five patients were in hematologic remission (CR) with minimal residual disease (MRD+), one patient was refractory to imatinib. Four of the five MRD+ patients are alive in CR after a median treatment duration of 20 (11-21) months. Two of these patients are in continuous CR 21 months after imatinib was initiated, while the other two patients experienced an isolated meningeal relapse that was successfully treated with additional intrathecal chemotherapy. Sustained molecular remissions were achieved in three patients and are ongoing 13 and 10.5 months after central nervous system (CNS) relapse and 6 months after starting concurrent IFN-alpha and imatinib, respectively. Marrow relapse occurred in one of the five MRD+ patients. Combination treatment was associated with a complete marrow response of 5 months duration in the imatinib-refractory patient. Imatinib combined with low-dose IFN-alpha may achieve prolonged hematologic and molecular remissions in a subset of patients with advanced Ph+ALL, who are not candidates for allogeneic SCT. CNS prophylaxis is necessary and may enhance the antileukemic activity of imatinib and IFN-alpha.

Unmodified phosphodiester antisense oligodeoxynucleotides to the BCR-ABL junction do not suppress Philadelphia-positive clonogenic cells.

The BCR-ABL fusion gene is directly involved in the pathogenesis of chronic myeloid leukemia (CML). Specific inhibition of the BCR-ABL gene expression with antisense oligodeoxynucleotides has been shown to have profound effects on cell growth in vitro. We examined antisense phosphodiester oligonucleotides (16-mers at a concentration of 60 micrograms/ml) spanning the two possible junction sites K28 (b3a2) and L6 (b2a2) in a clonogenic assay. Single colonies from 9 patients with CML and from patients with normal bone marrow were screened for BCR-ABL expression with a new 'single-tube nested PCR' method. There was a marked reduction in colony number in the CML group and a lesser growth inhibition in the control group. The number and percentage of BCR-ABL-positive colonies, however, was not reduced in the CML group. This indicates a nonspecific growth inhibition only.

Detection of fms-oncogene-specific tyrosine kinase activity in human leukemia cells.

The c-fms protooncogene encodes the receptor for the colony-stimulating factor 1 of macrophages. Its transforming counterpart, the v-fms oncogene has previously been recognized as the transforming gene of the McDonough strain of feline sarcoma virus. We have isolated rabbit antisera against a 115-kDa recombinant polypeptide containing the 926 carboxy-terminal amino acids of the v-fms protein. All antibodies recognized the cytoplasmic domain of the v-fms protein, which is 95% homologous to the corresponding domain of human c-fms proteins. These sera were applied in a survey of various human cancer cell lines, such as peripheral blood mononuclear (HL60) and choriocarcinoma (BeWo) cells, as well as leukemic cells from 58 patients with acute myelocytic, chronic myelocytic or acute lymphocytic leukemias (AML, CML, ALL). Significantly enhanced levels of fms-specific tyrosine kinase activity were detected in 12-O-tetradecanoylphorbol-13-acetate-induced HL60 and in BeWo cells, and in 7 out of 24 samples from AML patients, whereas no activity could be detected in 9 ALL or in 25 CML cell preparations. The AML cells were classified according to the FAB criteria. The highest incidence of increased fms activity was found in cells assigned to the M4 class (four out of five cases). While no activity was found in material belonging to FAB classes M2 or M3, one of the two cases of the M5 class was kinase-positive. Interestingly, two out of seven cases of the M1 class cells exhibited enhanced levels of fms kinase. These data suggest that the determination of the fms kinase may be useful to subdivide the M1 class of the FAB classification into monocytic and non-monocytic precursor leukemia cells.

Appropriate glycosylation of the fms gene product is a prerequisite for its transforming potency.

Processing inhibitors of N-linked glycans were used to determine whether correct glycosylation of the oncogene product gp140v-fms, encoded by the McDonough strain of feline sarcoma virus (SM-FeSV), is required to maintain the oncogenic properties of v-fms. SM-FeSV-transformed cells treated with the glucosidase-I inhibitors N-methyldeoxynojirimycin (MdN) or castanospermine synthesized predominantly a gp125v-fms species which had a normal half life. The molecule was transported to the plasma membrane and exhibited normal kinase activity as determined by autophosphorylation. However, although no significant change in cell morphology of the SM-FeSV-transformed cells was observed in the presence of castanospermine, growth of these cells became strictly serum-dependent. In addition, growth in soft agar was drastically retarded despite the presence of 10% calf serum, indicating that the transformed properties of the cells were altered. In contrast, swainsonine, an inhibitor of the processing alpha-mannosidase-II, had no effect. Cells transformed by the Snyder Theilen strain of FeSV were used to demonstrate that the altered proliferative properties were directly linked to the modified structure of the fms gene product. Our data suggest that the extracellular domain of gp140v-fms plays a role in regulating cell proliferation.

Purification and characterization of the feline sarcoma virus tyrosine-specific kinase pp85gag-fes.

The transforming phosphoprotein pp85gag-fes of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) was purified in a form which exhibits tyrosine-specific kinase activity. Cell lysates of ST-FeSV-transformed mink nonproducer cells were applied to a column of DEAE-Sephacel and eluted with a linear gradient of NaCl in phosphate buffer. Kinase activity was found uncomplexed (pp85gag-fes) in the flow-through and was eluted with 200 mM NaCl in a complex with pp50 and pp90. The flow-through material was further fractionated by chromatography on hydroxylapatite, followed by phosphocellulose and a final gel filtration step using Sephacryl S200. The final preparation was specifically enriched 1400-fold, and tyrosine-specific kinase activity was increased by about 300-fold as determined by autophosphorylation or phosphorylation of casein. Pp85gag-fes kinase activity was inhibited by nicotinamide adenosine dinucleotide (NAD) and slightly inhibited by NADH, while quercetin, a strong inhibitor for pp60src-associated tyrosine kinase activity, had no inhibiting effect.

Determination of phosphoethanolamine in urine or in the presence of high taurine concentrations.

Phosphoethanolamine forms a low-solubility lead salt, which may be precipitated from urine together with inorganic phosphate, thus separating taurine, urea, and other interfering substances of chromatographic determination. Fluorometric detection of phosphoethanolamine with o-phthaldialdehyde after elution from a Dowex 50-X10 column enhances the specificity of the method once more. The method described is fast and easy to perform.

Adult hypophosphatasia without apparent skeletal disease: "odontohypophosphatasia" in four heterozygote members of a family.

Twenty members of a family with adult hypophosphatasia were examined clinically and biochemically. Severe caries causing early loss of permanent teeth was the only clinical symptom which could be attributed to hypophosphatasia. None of them had a history of defective bone mineralization, rachitic skeletal alterations, and recurrent pseudofractures or fractures. An iliac crest bone biopsy of the proposita showed a normal finding corresponding to the age of the patient. Four family members in two subsequent generations were affected, thus suggesting an autosomal dominant inheritance. Their serum and leukocyte alkaline phosphatases were reduced. The phosphoethanolamine (PEA) excretion in the urine was increased to a level which suggests a heterozygote state. The serum alkaline phosphatase activity could be ascribed to the liver isoenzyme fraction. This was shown by polyacrylamide electrophoresis, by inhibition studies with organ-specific inhibitors, heat inactivation, inhibition by antibodies, and treatment with neuraminidase. The proposita had an unexplained, diffuse fatty infiltration of the liver. Thus, not only alterations of bone but also of liver metabolism in hypophosphatasia should be considered. The variety of adult hypophosphatasia described in this paper is characterized by the lack of severe bone abnormalities, the apparently autosomal dominant inheritance, and the reduction of bone and intestinal isoenzyme in the serum. Our study suggests that hypophosphatasia is a heterogeneous disorder which includes both severe and clinically mild forms.