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A method employing CD45-gating of blast cells was evaluated for the flow cytometric detection of residual disease in CD19- and myeloid antigen-positive acute leukemia in morphologic remission. In the normal bone marrow, CD45-gating identified at least three populations of immature cells, one of which appeared to retain a minute fraction of CD19- and CD13/33-antigen co-expressing cells. In acute leukemia, CD45 expression separated the blast cell population from the normal marrow cell populations. In the majority of patients with CD19- and myeloid antigen-positive acute leukemia, subpopulations of blast cells with this mixed phenotype were detected during morphologic remission.

We used fluorescence DNA in situ hybridization (FISH) to detect chromosomal abnormalities as an indicator of minimal residual disease in follow-up samples from the bone marrow (BM), or peripheral blood, of 25 patients with leukemia, lymphoma and myelodysplastic syndromes. Trisomies were detected by interphase FISH with repeat-sequence probes (RSP) or by using metaphase FISH with whole-chromosome paint probes (WCP). Specific translocations were detected using WCP probes. Translocations were observed using metaphase FISH in two patients in uncertain or complete remission (CR), who both later suffered relapse. Five patients with no abnormal cells remained in CR. Four patients with trisomies detected during CR suffered relapse; metaphase FISH detected the trisomy in 0.17-16% of metaphase cells. Five patients for whom the trisomy occurred in 0.034% of cells remained in CR. Trisomic nuclei were observed in 0.27-2.3% of interphase cells, by means of RSPs, in four patients who later suffered relapse. Five patients with trisomic nuclei in 0.061% remained in CR. When two probes were used simultaneously in a sample from one patient, 1% of the residual cells were abnormal. The patient later suffered relapse. In one patient with anaplastic large cell lymphoma, CD30-positive interphase cells were shown to have trisomic chromosome 7 by immunophenotyping and FISH. Our results suggest that metaphase FISH using WCP probes is a sensitive and specific method for detecting minimal residual disease especially in patients with translocations.

Alterations in DNA methylation appear to be an integral part of the malignant transformation. For example, the p15 region of chromosome 11 with multiple genes related to cell growth regulation exhibits different methylation patterns in the 5' area of the calcitonin A gene in healthy bone marrow cells, and in leukemic cell populations. In this work the methylation status of the 5' area of the calcitonin gene in myeloproliferative disorders (MPD) other than chronic myeloid leukemia (CML) is studied. A total number of 37 patients with polycythemia vera, essential thrombocythemia, or myelofibrosis were studied. A control group of 18 healthy persons and patients with reactive hematologic changes was included. The DNA isolated from peripheral blood or bone marrow cells was digested with the methylation-sensitive HpaII restriction enzyme. A Southern blot was hybridized with a 1.7 kb probe specific to the 5' area of the calcitonin gene. The result was visualized autoradiographically and analyzed with a densitometer. The results have been expressed as ratios between the abnormal and normal autoradiography band intensities, referred to as the calc-value or CALC. An increase in the calc-value signifies increasing methylation. In the control group the calc-value had a mean of 0.274. The myelofibrosis patients exhibited very strong hypermethylation in the calcitonin gene 5' area, with a mean calc-value of 11.1 (median 2.6). The polycythemia vera patients showed considerable variation in their methylation status, with a mean value of 1.52. The essential thrombocythemia patients exhibited weak hypermethylation, with a mean calc-value of 0.58. A correlation between karyotypic abnormalities and hypermethylation was observed. Complicated forms of MPD exhibited higher levels of methylation than the uncomplicated disease forms.

In order to study the efficacy of an oral induction and consolidation regimen in the treatment of acute myeloid leukemia (AML) in elderly patients assessed not to tolerate full-scale intensive chemotherapy, 51 patients over 65 years of age with newly diagnosed AML were randomized to receive two cycles of either totally oral ETI (25 patients) or conventional 5-day TAD (26 patients). The median age of the patients was 73 years, range 65-87 years. Thirty-eight patients had de novo AML and the remaining patients AML subsequent to myelodysplastic syndrome ((n = 11) or treatment related AML (n = 2)). ETI consisted of etoposide 80 mg/m2 and thioguanine 100 mg/m2 twice a day on days 1-5, and idarubicin 15 mg/m2 on days 1-3, all given orally. TAD consisted of oral thioguanine and i.v. cytarabine, both in the dose of 100 mg/m2 twice a day on days 1-5, and daunorubicin 60 mg/m2 on day 5. The maintenance treatment was daily oral mercaptopurine 70 mg/m2 and weekly oral methotrexate 12 mg/m2. In the ETI group complete remission (CR) was achieved in six patients after the first cycle and in nine more patients after the second cycle. The CR rate was 15/25 = 60%. The corresponding figures for the TAD group were four and two remissions, CR rate 6/26 = 23% (p = 0.007). The survival was significantly longer in the ETI arm (p = 0.042). The median survival was 9.9 months in the ETI group and 3.7 months in the TAD group. There were no significant differences in the side effects between the two arms. In conclusion, the totally oral ETI regimen resulted in a significantly higher remission rate and longer survival than the 5-day TAD regimen in elderly patients with AML, with no more toxicity.

Chromosomal analysis is routinely used in follow-up studies of acute leukemia, but it cannot be used in patients who do not have karyotypic abnormalities in blastic phase (10-46% of all cases). Recently we have demonstrated that unspecific DNA-fingerprint (DNA-F) changes can be detected in blastic phase of leukemia by DNA-F analysis. These changes can be used as molecular markers of the disease and in the follow-up studies of acute leukemia karyotyping and DNA-F analysis are complementary. The comparative analyses of leukemia samples with both these methods could help to localize minisatellite loci in chromosomes and reveal new DNA areas related to leukemia. New, more specific probes targeted to specific chromosomes could consequently be developed. We compared the DNA-F to the karyotypes of 50 acute leukemia patients. In blastic phase, 19 patients had DNA-F alterations and 31 patients abnormal karyotypes, 12 patients had both DNA-F alterations and karyotypic abnormalities. DNA-F changes were detected in six of the 16 patients with normal karyotypes and 19 patients with normal DNA-F had abnormal karyotypes at the time of diagnosis. Eleven patients showed neither DNA-F changes nor abnormal karyotypes in blastic phase. Three acute myeloid leukemia (AML) patients with DNA-F changes in blastic phase had trisomy eight as a sole abnormality or combined with balanced translocation, which suggests that there might be AML associated minisatellite locus in chromosome 8. In other cases the karyotypes were complicated and no clear evidence of the relationship of DNA-F changes with other chromosomes could be found.

Novel techniques were used to detect which cell lineages were affected by monosomy 7 in a patient who had myelodysplastic syndrome and later developed acute leukemia. The patient had had paroxysmal nocturnal hemoglobinuria for 20 years before developing refractory anemia with excess of blasts. Cytogenetic analysis at the myelodysplastic stage disclosed monosomy 7 in bone marrow mitoses. Restriction fragment length polymorphism analysis of fractionated white blood cells with the chromosome 7-specific probes MetH and MetD revealed that blood monocytes and most bone marrow erythroblasts but not blood granulocytes or lymphocytes were affected by monosomy 7. The patient later developed acute myelomonocytic leukemia with blast cells positive for markers of the myelomonocytic lineage but negative for granulocytic markers in a standard surface marker analysis. The leukemic blast cells had monosomy 7 as determined by direct cytogenetic investigation. Thus, the monocytes were found to be affected by monosomy 7 in this patient 8 months before her myelodysplastic syndrome progressed to acute myelomonocytic leukemia, and the affected cells had the same biologic markers at both stages of the disease.