Sanford A. Stass

Preliminary results of treatment with filgrastim for relapse of leukemia and myelodysplasia after allogeneic bone marrow transplantation

BACKGROUND Patients whose leukemia relapses after allogeneic bone marrow transplantation have a poor prognosis; few respond to further chemotherapy, and almost none survive over the long term. We present preliminary observations on the use of filgrastim (granulocyte colony-stimulating factor) for relapse after transplantation. METHODS Seven female patients with leukemia (one with chronic myelogenous leukemia, five with acute myelogenous leukemia, and one with a myelodysplastic syndrome that transformed into acute myelogenous leukemia) whose disease relapsed within 360 days after allogeneic bone marrow transplantation received filgrastim (5 micrograms per kilogram of body weight per day by subcutaneous injection) to reinduce remission by stimulating residual donor marrow cells. Cytogenetic analysis of bone marrow, fluorescence in situ hybridization, and determination of restriction-fragment--length polymorphisms were used to assess response and chimerism. RESULTS Three of the seven patients had a complete hematologic and cytogenetic remission, with reestablishment of hematopoiesis of donor origin. Mild chronic graft-versus-host disease developed in one patient, and acute graft-versus-host disease in none. One patient had a relapse 12 months after treatment, and two others remained in remission after 10 and 11 months. In two of the patients with a response, fluorescence in situ hybridization demonstrated stimulation of donor cells without differentiation of the leukemic clone. CONCLUSIONS Filgrastim may be effective in selected cases of leukemic relapse after allogeneic bone marrow transplantation.

Philadelphia chromosome-negative chronic myelogenous leukemia with rearrangement of the breakpoint cluster region. Long term follow-up results

Background. Five to 10% of patients with chronic myelogenous leukemia (CML) do not have the Philadelphia chromosome (Ph), but one‐third of them have rearrangements of the breakpoint cluster region (BCR‐positive).

All-trans retinoic acid followed by chemotherapy for salvage of refractory or relapsed acute promyelocytic leukemia

Background. All‐trans retinoic acid (ATRA) is effective in the treatment of relapsed or refractory acute promyelocytic leukemia (APL), but relapse is the rule if response is unmaintained.

Significance and correlations of molecular analysis results in patients with philadelphia chromosome-negative chronic myelogenous leukemia and chronic myelomonocytic leukemia

PURPOSE Several investigators have documented a rearrangement of the breakpoint cluster region (bcr) in selected patients with a morphologic diagnosis of chronic myelogenous leukemia (CML) but no abnormality of the Philadelphia chromosome (Ph) by cytogenetic studies. Our intention was to systematically investigate the incidence of the bcr rearrangement in such patients, and to correlate the findings with patient characteristics, response to therapy (especially alpha interferon treatment), and overall prognosis. PATIENTS AND METHODS Molecular analysis studies were performed in 40 patients with Ph-negative CML (23 patients) and myelomonocytic leukemia (CMML; 17 patients). RESULTS Rearrangement of the breakpoint cluster region (bcr) was detected in 11 of the 23 patients with Ph-negative CML (48 percent), indicating the presence of the abnormal molecular events in Ph-positive CML without documentation of the Ph cytogenetic abnormality. None of the 17 patients with CMML had the bcr rearrangement. Patients with Ph-negative CML and the bcr rearrangement had characteristics similar to those of patients with Ph-positive disease. These included a younger age, higher white blood cell counts, a higher incidence of thrombocytosis and basophilia, and a lower occurrence of thrombocytopenia. The leukocyte alkaline phosphatase score was not a helpful distinguishing feature. Among 21 patients receiving alpha interferon-based regimens, response to therapy was significantly better among patients with Ph-negative disease and the bcr rearrangement (seven of seven, 100 percent), compared with those without the bcr rearrangement (one of six, 17 percent), or patients with CMML (two of eight, 25 percent) (p less than 0.01). At this time of follow-up, only one of the 11 patients with Ph-negative CML and the bcr rearrangement had died from complications of allogeneic bone marrow transplantation, compared with three deaths among the 12 patients with Ph-negative CML and no bcr rearrangement, and 11 deaths among the 19 patients with CMML. CONCLUSION We conclude that molecular studies help in better understanding the nosology of Ph-negative CML, and define a subgroup of patients with clinical, therapeutic, and prognostic correlations similar to those of patients with Ph-positive CML.

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.

DNA aneuploidy in adult acute leukemia

Using flow cytometric techniques, we determined DNA ploidy levels in the bone marrow of 318 successive adult patients with newly diagnosed acute leukemia. Overall, 26% exhibited DNA stem line abnormalities, usually with a 10%-15% DNA excess, regardless of morphologic diagnosis. DNA aneuploidy was seen most frequently in patients with a hyperdiploid chromosome number and karyotype instability (50%), but was also present in a third of patients with chromosomal translocations and in 20% of patients with a normal diploid karyotype. Thus, among 73 patients with DNA aneuploidy, quantitatively concordant karyotype abnormalities were observed in almost 40% of patients; the discrepancy between DNA content and chromosome number in the remaining patients may reflect differences in the cell cycle position of target cells in G1/0 phase or mitosis, respectively. Cytogenetics affected treatment outcome in acute myelogenous leukemia (AML) with more favorable short- and long-term prognosis among patients with translocations compared with those with numeric abnormalities. The presence of an abnormal DNA stem line, among AML patients with translocations, identified a favorable subgroup with significantly longer remission duration and survival (25 and 26 months versus 18 and 13 months, respectively). In addition, the prognostic implications of DNA aneuploidy in AML were age-dependent, in that favorable effects among patients with translocations and unfavorable effects among those with numeric abnormalities or diploid karyotypes were most obvious in young and not in older patients (greater than or equal to 40 years). In adults with ALL, DNA aneuploidy was associated with shorter survival (15 versus 39 months in the diploid group), an observation that is distinctly at variance with recent findings in childhood ALL. Our results indicated that DNA flow cytometry was complementary to standard cytogenetics for the detection of genomic abnormalities; and DNA aneuploidy emerged, like in children but not in adults with ALL, as a new favorable prognostic feature in a subgroup of adults with AML, the biologic basis of which remains to be determined.

Expression of differentially phosphorylated Rb and mutant p53 proteins in myeloid leukemia cell lines.

We studied the structure and expression of Rb and p53 genes in six myeloid leukemia cell lines (HL-60, KBM3, K562, KBM5, EM2, KBM7) in the light of the published reports that structural abnormalities of these genes are rarely seen in leukemic cells and also a recent finding that Rb gene expression can be regulated by the p53 protein. Except for HL-60 cells which have a truncated p53 gene, none of the other cell lines revealed any gross structural abnormalities in the Rb and p53 genes. KBM3, KBM5 and EM-2 expressed lower levels of Rb mRNA than HL-60, K562 and KBM7. The amount of Rb protein was lowest in KBM3 cells and in this and two other cell lines (KBM5, KBM7) Rb was markedly hypophosphorylated compared to the other three cell lines. HL-60 and K562 did not express p53 m-RNA, while the other four cell lines all expressed high levels of mutant p53 protein. Thus even in the absence of gross structural alterations, subtle abnormalities in the expression pattern of Rb and p53 genes occur in myeloid leukemia cells.