Lymphotoxin is an autocrine growth factor for Epstein-Barr virus-infected B cell lines.

Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)

Definition of the high-risk acute lymphoblastic leukemia patient by immunological phenotyping with monoclonal antibodies.

An accurate method of classification of the surface membrane characteristics of blast cells from patients with acute lymphoblastic leukemia would allow a more definitive study of the nature of this disease. Monoclonal antibodies have been produced to the surface antigens of leukemic blasts form a patient with high-risk acute lymphoblastic leukemia. Two antibodies of interest were obtained from this immunization. These two, in combination with a monoclonal antibody with anti-Ia specificity, have been used to obtain surface phenotypes for patients with childhood acute lymphoblastic leukemia. Preliminary results indicate that the definition of a high-risk group, using these antibodies, possible.

Heterogeneity in lineage derivation of Philadelphia-positive acute lymphoblastic leukemia expressing p190BCR-ABL or p210BCR-ABL: determination by analysis of individual colonies with the polymerase chain reaction.

The molecular hallmark of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) is the expression of 1 of 2 alternate forms of the aberrant BCR-ABL protein-p210BCR-ABL or p190BCR-ABL. The presence of BCR-ABL message provides a target for analyzing the lineage derivation of this disease. We, therefore, studied myeloid and erythroid progenitor involvement in Philadelphia chromosome-positive ALL. Bone marrow low-density cells from Philadelphia chromosome-positive ALL patients (5 with the p190BCR-ABL and 2 with the p210BCR-ABL anomaly) were cultured in the mixed colony culture assay. cDNA from individually plucked colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid colonies was then analyzed using the hybridization protection assay in conjunction with the polymerase chain reaction to detect BCR-ABL molecular aberrations. Colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid colonies from 1 of 5 p190BCR-ABL-positive patients and 1 of 2 p210BCR-ABL-positive patients expressed BCR-ABL transcripts, whereas colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid colonies from the other patients did not. Our study suggests that the origin of both p190BCR-ABL- and p210BCR-ABL-positive ALL is heterogenous with involvement of either a pluripotent precursor or a lymphoid lineage-committed hematopoietic progenitor.

Phorbol ester effects on topoisomerase II activity and gene expression in HL-60 human leukemia cells with different proclivities toward monocytoid differentiation.

We examined the effects of phorbol ester treatment on topoisomerase II-mediated events in two human leukemia cell lines with different proclivities toward phorbol ester-induced monocytoid differentiation. HL-60 is the parent line that will terminally differentiate; 1E3 is a derived line that will not terminally differentiate. Within 24 h of phorbol ester treatment, etoposide-induced, topoisomerase II-mediated DNA cleavage declined 10-fold, whereas 4'-(9-acridinylamino)-methanesulfon-m-anisidide- induced DNA cleavage declined 3-fold in HL-60. In phorbol-treated 1E3, etoposide-induced DNA cleavage declined only 2-fold, whereas 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced cleavage was barely affected. There was a 2- to 3-fold decline in topoisomerase II activity within the nuclear extracts from phorbol-treated HL-60 cells but not from phorbol-treated 1E3 cells. Immunoblotting experiments with anti-topoisomerase II antibodies indicated that phorbol treatment produced a structural change in the immunoreactive topiosomerase II in HL-60 nuclear extracts but produced no change in 1E3 topoisomerase II. Phorbol ester treatment also produced a decline in the level of topoisomerase II gene expression in HL-60 but not in 1E3 cells. By contrast, the cytotoxicity of etoposide in both lines was decreased following phorbol treatment. Thus, phorbols may uncouple the mechanisms linking drug-induced, topoisomerase II-DNA cleavable complex stabilization with drug-induced cytotoxicity, particularly in 1E3.

Modulation of a constitutive transcriptional block at exon-1 controls human c-myc oncogene expression.

C-myc gene down-regulation is known to be mediated by a transcriptional block at the end of exon-1 (Bentley & Groudine, 1986; Siebenlist et al., 1988). When transcription is initiated normally, this block is expected to produce a truncated RNA, but to date, this product has escaped direct detection in somatic cells. We have been able to detect a 0.38 kb c-myc exon-1 specific RNA species by northern blot analysis. This RNA appeared not only in dimethyl sulfoxide (DMSO)-induced HL-60 cells, but also in uninduced HL-60 cells, the NALM-6, REH, RPMI-8392 and TALL-1 cell lines, and in human T-cell acute lymphocytic leukemia (T-ALL) and normal peripheral blood lymphocytes (PBL), indicating that the transcriptional block producing it is constitutive. In HL-60 cells, the 0.38 kb RNA level increased on DMSO induction while the 2.3 kb c-myc mRNA was down-regulated. Upon DMSO removal, as the 2.3 kb mRNA was made again, the 0.38 kb RNA fell to preinduction levels, Thus, modulation of this constitutive block regulates the relative amounts of c-myc message available for translation in response to specific growth stimuli. This mechanism for c-myc gene regulation may be common within the hematologic system.

Accurate quantitation of residual B-precursor acute lymphoblastic leukemia by limiting dilution and a PCR-based detection system: a description of the method and the principles involved.

The detection of residual leukemia cells in the bone marrow of patients during morphologic remission has been greatly facilitated by use of the polymerase chain reaction (PCR) to amplify leukemia-specific sequences. While the current PCR strategies for estimating the amount of residual leukemia claim a detection sensitivity of one leukemia cell amongst 10(5) or 10(6) normal cells, a rigorous assessment of the relative error associated with these techniques has not been presented. We have developed a method of estimating the amount of residual leukemia in remission marrows that is analogous to the limiting dilution assays used to determine the frequency of immunocompetent cells in a responder cell population. Using this method we measured the fraction of all-or-none (i.e. positive or negative) reactions of the PCR amplification of the leukemia-specific IgH gene rearrangement in replicate samples of serial dilutions of DNA obtained from diagnostic bone marrow specimens from 15 children with B-precursor acute lymphoblastic leukemia (ALL). A sigmoid curve representing the fraction of positive PCR reactions at a given dilution of leukemia DNA was found to be the best fit to the data. The narrowness of the log-linear region of this curve prevents the direct application of the analysis methodology that has previously been described for limiting dilution assays. However, the residual leukemia burden during morphological remission in these 15 patients and in two additional patients who experienced relapse could be estimated by the described dilution analysis method using the best-fit equation. Furthermore, the data generated for diagnostic, remission and relapse marrow samples exhibited a small interspecimen variation. The results suggest that this method can reliably estimate residual leukemia over a range of five orders of magnitude. Although the PCR reaction appears to be one of the most sensitive methods for detecting residual leukemia, all techniques based on this procedure, including our own, must exhibit limitations inherent to the amplification process. Our estimates or relative error suggest that a realistic limit for the PCR estimation of residual leukemia lies in the range of one leukemia cell per 10(5) normal cells. The suggested method is rapid, technically simple and relatively inexpensive. Furthermore, the principles that it is based upon can be applied to any PCR-based strategy.

Inhibition of acute myelogenous leukemia progenitor proliferation by macrophage inflammatory protein 1-alpha.

Macrophage inflammatory protein-alpha (MIP-1 alpha), an 8-kDa peptide produced by stimulated macrophages, has been recently sequenced and cloned. In addition to its inflammatory effects, MIP-1 alpha inhibits proliferation of immature hematopoietic progenitors both in vitro and in vivo. Because the gene coding for MIP-1 alpha is expressed in peripheral blood cells obtained from patients with acute myelogenous leukemia (AML), we sought to evaluate the effect of MIP-1 alpha on AML precursors. We studied bone marrow samples from 21 AML patients using both the AML blast colony assay and the delta suspension culture assay. We found that recombinant human (rh) MIP-1 alpha significantly inhibits early and mature AML progenitors with sample-to-sample variability, by up to 79% at concentrations ranging from 40 to 1600 ng/ml. These results were obtained in the presence of fetal calf serum either alone or with granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, or interleukin-3. In contrast, rhMIP-1 alpha (400 ng/ml) did not significantly affect normal colony-forming unit granulocyte-macrophage (CFU-GM), or burst-forming unit-erythroid (BFU-E) proliferation. These data prompted us to delineate the inhibitory mechanism of MIP-1 alpha. Consequently, we used the thymidine suicide technique to measure DNA synthesis in AML progenitors and the enzyme-linked immunosorbent assay to quantify intracellular levels of interleukin-1 beta in AML blasts following incubation with MIP-1 alpha. We found that whereas MIP-1 alpha prevented AML progenitors from entering the proliferative phase of the cell cycle, it had no effect on interleukin-1 beta levels. Taken together, our data suggest that MIP-1 alpha may have clinical benefits in therapy for AML and should be considered for evaluation in a clinical setting.

T cell receptor gene rearrangements in B-precursor acute lymphoblastic leukemia correlate with age and the stage of B cell differentiation.

Children with ALL diagnosed at less than 2 years of age have a poor prognosis when compared with older children. In an effort to identify biologic features of ALL in children less than 2 that might explain this difference, we performed extensive immunophenotypic and molecular genetic analyses on a series of patients. For comparison purposes patients were divided into four groups: CALLA- (CD10-) infants less than 2 years of age at diagnosis (n = 10), CALLA- children greater than 2 years of age at diagnosis (n = 10), CALLA+ infants (less than 2 years, n = 21) and CALLA+ children (older than 2 years, n = 21). No immunophenotyping differences in CALLA- or CALLA+ subgroups were identified when cases less than 2 were compared with cases greater than 2 years of age at diagnosis. The most interesting results were in the CALLA- group where 94% of the samples expressed the B cell antigen CD19 but 27% co-expressed CD7. Double labeling experiments confirmed leukemic blast cells co-expressed CD19 and CD7. The double-labeled cells represent either leukemic conversion of a precursor cell which has not yet committed to B or T cell lineage or aberrant expression of these antigens. Molecular genetic studies demonstrated that all samples, regardless of the patients' age or immunophenotype, had rearrangement of the Ig heavy chain gene. The most striking molecular results were in CALLA- patients; in patients less than 2 at diagnosis neither the beta- nor the gamma-chain gene of the T cell receptor (TCR) was rearranged, whereas DNA from 5 of 10 patients over the age of 2 demonstrated beta- or gamma-chain TCR gene rearrangements. The percentage of CALLA+ cases under the age of 2 years with rearrangements in TCR genes is less than that found in CALLA+ cases over the age of 2 years. The finding of no TCR rearrangements in CALLA- ALL and a decreased number of gamma-TCR rearrangements in CALLA+ cases under the age of 2 suggest that age may affect TCR gene rearrangements in lymphoblasts. The molecular differences in TCR gene rearrangements do not appear to correlate with the response to therapy.

Persistence of self-renewing leukemia cell progenitors during remission in children with B-precursor acute lymphoblastic leukemia.

No effective therapy is available for the majority of the 30-40% of children with acute lymphoblastic leukemia (ALL) who relapse. Since the morphologically undetectable, or occult, leukemia cells that persist during remission originate from the clone present at diagnosis, may also have both the capability to sustain the disease and to give rise to relapse, we are evaluating a method of identifying them. We have combined, for the first time, an ALL blast colony assay (BCA) and the polymerase chain reaction (PCR) to isolate residual leukemia cells in remission bone marrow aspirate specimens from eight patients with B-precursor ALL during early continuation therapy. We found colony-forming leukemia cells with in vitro self-renewal capability that survived chemotherapy for 15 months after diagnosis in all sequential specimens from these patients. To verify the leukemic nature of these cells their DNA was amplified by PCR and the product directly sequenced. In every case, the VHDJH sequence observed at diagnosis was found. None of the patients relapsed during this early phase of their treatment, consistent with the observation that patients with B-precursor ALL experience recurrence late in their course. Since it is possible that some of these persistent leukemia cells belong to the leukemia progenitor cell population that sustains the disease, the study of them could provide the means to determine the mechanisms of relapse.

Myeloid surface antigen-positive acute lymphoblastic leukemia (My+ ALL): immunophenotypic, ultrastructural, cytogenetic, and molecular characteristics.

Leukemic blasts from 40 consecutively admitted adults with untreated acute lymphoblastic leukemia (ALL) were examined for myeloid surface antigen expression. Of these, 14 (35%) were reactive with one or more myeloid monoclonal antibodies. Each example of myeloid surface antigen-positive (My+ ALL) met the standard morphologic and cytochemical criteria for ALL. In addition, none of the 13 samples studied for ultrastructural evidence of myeloperoxidase met the criteria for acute myelocytic leukemia (AML). All patient samples reacted with lymphoid monoclonal antibodies: CD10+ (8 patients), CD19+ CD10- (2 patients), T cell+ (2 patients), and T cell+ CD10+ (2 patients). Coexpression of myeloid and lymphoid determinants was established by two-color immunofluorescence studies using flow cytometry in five of five samples analyzed. Cytogenetic abnormalities that have been associated with myeloid and mixed leukemias were common, including t(9;22), 7q-, abnormalities of 11q with or without a translocation, 20q-, and -5. Blasts from seven patients were studied at the molecular level. Immunoglobulin heavy chain gene rearrangements were detected in five of five samples with B cell+ T cell- phenotypes. One sample that was T cell+ CD10+ was germline for the immunoglobulin heavy chain and the T cell receptor gamma- and beta-chain genes. The other patient with T cell+ CD10+ blasts relapsed with AML following allogeneic bone marrow transplantation. The leukemia cells at the time of diagnosis and the cells at relapse demonstrated similar cytogenetics and the same immunoglobulin gene rearrangement, suggesting a clonal relationship. As a group, the My+ ALL patients had a significantly decreased complete remission rate when compared to My- ALL patients. Further studies at the molecular level will be required to determine the significance of karyotype abnormalities in My+ ALL.

Comparison of the 40 kDa hematopoietic cell antigens bound by monoclonal antibodies IV.3, 41H.16 and KB61.

A study is presented which compares the properties of antigens with relative mol. mass of about 40,000, detected by three different monoclonal antibodies: IV.3, 41H.16 and KB61. Antibody IV.3 has been shown (in work by other investigators) to detect the Fc receptor for IgG. It recognizes this molecule on neutrophils, macrophages and platelets. Antibody 41H.16 precipitates a molecule of mol. wt of approximately 40,000, which is present on B cells, neutrophils and macrophages. The antigens precipitated by IV.3 and 41H.16 have been compared by isoelectric focusing, sequential antibody-mediated affinity chromatography and trypsin hydrolysis. The results indicate that these two antibodies bind different molecules of similar relative mol. mass. A third antibody, KB61, has been reported, which also reacts with a 40,000 mol. wt molecule and has a distribution of cellular reactivity which is similar to 41H.16. Sequential preclearing experiments have shown that these molecules recognize the same antigen.

Characterization of gp39, a B-lymphocyte associated differentiation antigen which is also present on granulocytes and macrophages.

The biosynthesis and biochemical characteristics of the 39,000 cell surface glycoprotein detected by Mab 41H.16 were investigated. Experiments utilizing tunicamycin, endoglycosidase H, endoglycosidase F and N-glycosidase F indicate that the mature molecule expressed at the cell surface is composed largely of N-linked oligosaccharides of both the complex and high mannose types. When synthesized in the presence of tunicamycin, the molecule appeared on the cell surface with a Mr of 32,000. Digestion with both endoglycosidase H and endoglycosidase F yielded a single band of Mr 37,000. Parallel experiments with N-glycosidase F revealed species of approx. 35,000 and 32,000. Synthesis in the presence of monensin yielded a 37,500 product. [3H]Glucosamine and [3H]mannose were incorporated into the molecule but no evidence for fucose incorporation could be found. Microheterogeneity of gp39 with respect to Mr and oligosaccharide structure was demonstrated by biosynthetic labelling and lectin chromatography. Biosynthetic pulse-chase labelling showed that the de novo synthesis of the 39,000 molecule occurs without detectable precursor formation. Results of temperature-dependent phase separation experiments were consistent with gp39 being an integral membrane protein. Two-dimensional electrophoresis showed heterogeneity of the isoelectric points associated with the N-linked oligosaccharides. Galactose oxidase/NaB[3H]4 labelling showed that a terminal sialic acid protects a galactose residue. All results are consistent with the conclusion that the gp39 molecule is an integral membrane glycoprotein composed of heterogeneous N-linked oligosaccharides of both the complex and high mannose types.

Phorbol regulation of topoisomerases I and II in human leukemia cells. Studies in an additional cell pair sensitive or resistant to phorbol-induced differentiation.

We previously reported (Zwelling et al., Cancer Res 50: 7116-7122, 1990) that etoposide-induced DNA cleavage and mRNA coding for topoisomerase II are reduced in HL-60 cells induced to differentiate by phorbol ester. Reduction of etoposide-induced cleavage and topoisomerase II message did not occur in the derived cell line 1E3 (which is resistant to phorbol-induced differentiation), implying that topoisomerase II activity may be related to the state of cell differentiation. We have extended these studies using a new phorbol sensitive/resistant cell pair, S (sensitive) and PET (phorbol ester tolerant). Phorbol ester exposure not only reduced etoposide-induced DNA cleavage and topoisomerase II mRNA in S cells but also decreased the amount of immunoreactive topoisomerase II enzyme in whole S cells. However, immunoreactive topoisomerase II extracted from the nuclei of phorbol-treated S cells was not reduced compared with that from the nuclei of untreated S cells. This suggests that topoisomerase II contained in nuclear extracts is not always representative of the total cellular enzyme. Dramatic decreases in the amount, activity, or gene expression of topoisomerase II were not observed after phorbol treatment of the resistant PET cells; this is consistent with the potential involvement of topoisomerase II in monocytoid differentiation. Levels of topoisomerase I enzyme and mRNA fell in both S and PET cells after phorbol treatment; therefore, the genes for topoisomerases I and II did not appear to be regulated coordinately.

Molecular genetic evidence for a differentiation-proliferation coupling during DMSO-induced myeloid maturation of HL-60 cells: role of the transcription elongation block in the c-myc gene.

Proliferation-differentiation coupling was studied during dimethyl sulfoxide (DMSO)-induced myeloid maturation of HL-60 cells using transcription of the myeloperoxidase (MPO) and c-myc genes as indicators of differentiation and proliferation, respectively. Concomitant cell cycle kinetic analysis correlated the proliferation and transcription patterns. Transcription, cell cycle phases and rate of DNA synthesis were examined for up to 5 days of induction and, at 1-day intervals, analyzed during a 24-h reculture without the inducer. DMSO suppressed transcription of the c-myc and MPO genes with a t1/2 of 16 min and 7 h, respectively. The ability to recover transcription following reculture diminished with the progression of the induction and ultimately was lost; concomitantly, the cells irreversibly lost the capacity to divide. This indicated that the differentiation and proliferation processes are inseparable and that terminal differentiation accompanies irreversible proliferation arrest in HL-60 cells. We also studied the kinetics of the block to transcription elongation at the exon 1-intron 1 boundary of the c-myc gene. This block produces a 0.38 kb truncated transcript that is constitutively expressed in somatic cells (Re et al., Oncogene 5, 1247, 1990). During induction the level of the 0.38 kb RNA increased, while that of the complete c-myc mRNA decreased, indicating that this truncated RNA is generated instead of message through a monotonously initiated transcriptional process. Transcription initiation and synthesis of the 0.3 kb RNA persisted in terminally differentiated cells, suggesting a role for this RNA in non-proliferating cells.

8;20 chromosomal translocation in a case of acute leukemia. Cytogenetic, immunophenotypic, ultrastructural, and molecular characteristics.

An 8;20 chromosomal translocation was observed in the leukemia cells of a 3-year-old girl. To our knowledge, this is the first report of this translocation in de novo acute leukemia. This chromosomal defect was present in the leukemia cells at diagnosis and also at relapse, but remission bone marrow cells had the 46,XX karyotype. By morphologic and cytochemical criteria the leukemia was myeloid but these features were more lymphoid when the leukemia recurred. However, the immunophenotype was consistent with myeloid leukemia and did not change at relapse. No evidence for either immunoglobulin or TCR gene rearrangement was observed.

Immature and differentiated neoplastic populations in acute lymphoid leukemia of childhood: biological and clinical implications.

Despite significant improvement in the therapy for acute lymphoid leukemia (ALL) of childhood, approximately 30% of patients relapse. Unfortunately, since no successful treatment for recurrent disease has been developed, the majority of these patients die. Recently, we presented evidence consistent with the presence of a limited program of differentiation in B-precursor ALL that is reminiscent of normal B-cell development. We found that ALL cell populations consist of both a subpopulation of progenitors with the immunophenotype of normal B-cell precursors that has self-renewal capability and a second subpopulation with a more mature early B-cell immunophenotype that is without self-renewal capability but can proliferate to a limited extent. In our recent studies we were able to grow the progenitor cells in the ALL blast colony assay and establish their leukemic origin using the polymerase chain reaction. Our results suggest that these progenitors are the cells that sustain the disease. We hypothesize that these cells may remain quiescent, for a time, and either eventually die or regain proliferative capability and cause relapse. Further studies aimed both at detecting residual ALL and determining changes in their biology may provide an understanding of the mechanisms of relapse in this disease.

The clinical significance of residual disease in childhood acute lymphoblastic leukemia as detected by polymerase chain reaction amplification by antigen-receptor gene sequences.

The polymerase chain reaction (PCR) has been applied to detect occult leukemia cells in children with acute lymphoblastic leukemia who are otherwise considered in complete remission by traditional morphological examination of bone marrow specimens. To determine whether PCR provides unique prognostic information of use for the clinical investigator, we reviewed the 20 clinical studies published to date. From this review, it is evident that discrepancies exist for the detection of residual disease for patients who remain in complete remission and for those who relapse. However, because of the fundamentally different approaches used to apply the PCR method to each of these studies, an entirely different interpretation can be reached when critical technical factors are considered. The combined data from the various studies suggest that a consistent pattern for residual disease disappearance over many months exists for patients who remain in extended complete remission and a pattern of residual disease persistence and reappearance preceding clinical findings exists for the majority of those who ultimately relapse in the bone marrow.

Detection of minimal residual disease in all: biology, methods, and applications.

The PCR technique appears to be the most sensitive method for detecting residual disease in ALL and can be applied to a high percentage of cases by amplifying sequences of the antigen-receptor genes. The PCR studies to date suggest that this sensitive technique can detect residual disease in virtually all patients during the first year of treatment. The residual disease becomes undetectable in the majority of patients by the end of treatment; however, a subset of patients remain PCR positive at a time when therapy is electively discontinued. The development of a highly accurate quantitative PCR technique may allow the possibility of distinguishing the patterns of residual disease for patients who will be cured by treatment from those who relapse. If such a pattern can be discerned, then an immediate benefit for PCR monitoring will be that clinicians will have the opportunity to test whether treating patients at the time of 'molecular relapse' will help to improve the cure rate for this disease. The PCR studies of remission marrows at the end of treatment raise a number of questions about the biology of disease persistence in patients who remain in extended 'remission.' A commitment to obtaining and analyzing bone marrow specimens in patients who have completed therapy is necessary to discern whether novel strategies, such as immunomodulatory manipulations, are needed to control or eradicated residual disease in patients who have completed planned chemotherapy. Thus, the long-term benefit of residual disease monitoring by PCR may be a better understanding of the biology and definition of 'cure' in ALL.

Chromatin structural analysis of the 5' end and contiguous flanking region of the myeloperoxidase gene.

Myeloperoxidase (MPO) synthesis is known to be associated with the promyelocyte stage of myeloid differentiation. In particular the downregulation of MPO gene transcription is associated with myeloid cell maturation. We examined the changes in the deoxyribonuclease I hypersensitive sites within the 5' end of the MPO gene and its 5' flanking region during dimethyl sulfoxide (DMSO)-induced differentiation of HL-60 cells to determine the changes in chromatin structure that accompany this process. The locations of hypersensitive sites surrounding the 5' end of the gene in proliferating, uninduced cells were determined: three were observed in the 5' flanking region and one within the gene. Progressive changes in all sites accompanied the downregulation of MPO transcription after treatment with DMSO. No evidence of hypersensitivity was observed in the chromatin region examined after 8 days of DMSO exposure. The results provide an example of the changes that occur in the chromatin structure of a gene as it is inactivated during differentiation.

A monoclonal antibody detecting a 39,000 m.w. molecule that is present on B lymphocytes and chronic lymphocytic leukemia cells but is rare on acute lymphocytic leukemia blasts.

Monoclonal antibody 41H.16 was produced by using the hybridoma methodology in the mouse system with cells from a patient with hairy cell leukemia as the immunogen. This antibody reacts with the majority of Slg+ cells in the peripheral blood and with all B lymphoblastoid cell lines. Reactivity with conventional Ig determinants, Fc or C3 receptors, has been excluded. The antibody reacted with cells from 68 patients with CLL but showed no reactivity with cells in 69 specimens from patients with non-T, non-B ALL. The apparent m.w. of the antigen detected by this antibody is approximately 39,000.