Leukaemias into the 21st century. Part 2: the chronic leukaemias.

Like the acute leukaemias, the chronic leukaemias are broadly classified according to their cell lineage of origin. Chronic myeloid leukaemia and chronic lymphocytic leukaemia are the most common disease entities within the myeloid and lymphoid lineages, although several less common entities are well recognised within each broad subgroup. In common with the dramatic progress in the acute leukaemias, there has been considerable progress in our understanding of the biology and molecular genetics of the chronic leukaemias that is now being translated into significant therapeutic advances.

Leukaemias into the 21st century: part 1: the acute leukaemias.

The leukaemias are a biologically and clinically heterogeneous group of malignancies, which manifest as clonal expansions of a single cell at different stages of lympho-haemopoietic development. The transformed cell acquires an unrestrained capacity for self-renewal and, in the case of the acute leukaemias, also fails to differentiate into functional mature cells. Historically leukaemias were classified using a combination of clinical and (presumed) cell lineage criteria. Thus, the four major subgroups of acute and chronic myeloid leukaemia and acute and chronic lymphoid leukaemia were recognised. Up until the last 10-15 years, patients within each major subgroup were treated along broadly similar lines. Genetic abnormalities have been recognised in certain leukaemias for over 50 years; however, the recent explosion in our understanding of the frequency and complexity of molecular abnormalities in the leukaemias has 'opened the door' for the design of more targeted therapies with the expectation that their incorporation into therapeutic regimens will be associated with greater efficacy and less off-target toxicity.

Clinical perspectives in lymphoma.

The classification of both Hodgkin's and non-Hodgkin's lymphomas continues to evolve. The current World Health Organization classification incorporates data derived from advances in our understanding of the pathogenesis of these disorders together with their distinguishing immunophenotypic, genotypic, clinical and histopathological characteristics. As outcomes have improved, the main emphasis of treatment has been to incorporate a risk-adapted approach to reduce long-term toxicity without sacrificing efficacy through the use of varying combinations of chemotherapy, radiotherapy and immunotherapy.

Paradoxical effect of BW 301U, a lipophilic antifolate, on methotrexate-inhibitable deoxyuridine incorporation by human hematopoietic cells.

The ability of methotrexate and BW 301U, a lipophilic folate antagonist, to inhibit tritiated deoxyuridine incorporation into acid-precipitable material by human bone marrow cells was evaluated before and after five sequential daily infusions of BW 301U. After in vivo BW 301U therapy, bone marrow cells from five of the six patients exhibited significantly reduced inhibition by 1 microM methotrexate in vitro, whereas the response to 1 microM BW 301U remained unchanged. Megaloblastic marrow morphology and decreased myeloid progenitor cloning efficiency were also observed following five daily BW 301U infusions of 21 and 71 mg/sq m, respectively. A similar reduction in the ability of methotrexate to inhibit tritiated deoxyuridine incorporation was also seen in HL-60 cells, a human acute promyelocytic leukemia cell line, after incubation in vitro with cytostatic concentrations of BW 301U for 3 days. Concomitant changes in the response to BW 301U did not occur. While it is premature to infer clinical significance from this preliminary observation of BW 301U-induced asymmetry in the response to subsequent antifolates, our results augment a growing body of evidence which suggests that lipophilic folate antagonists might be effective in the treatment of methotrexate-resistant neoplasms.

Characterization of the human N-ras promoter region.

Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1.

Allele-specific enrichment: a method for the detection of low level N-ras gene mutations in acute myeloid leukemia.

Point mutations involving codons 12, 13, and 61 of the N-ras gene are found in patients with acute myeloid leukemia (AML). We have developed a sensitive assay for the analysis of these mutations which we have called allele-specific enrichment. In this protocol the polymerase chain reaction (PCR) amplifies DNA with primers that introduce new restriction sites into the normal N-ras allele only. Digestion with the appropriate enzyme cleaves normal, but not mutant, alleles and this digested product provides a mutant allele-enriched template for a second round of amplification. The second PCR product is digested, Southern blotted and analyzed by allele-specific oligonucleotide (ASO) hybridization. This protocol is more sensitive than ASO hybridization alone and has revealed a minor clone in the DNA of a patient with AML. The method may be useful for the detection of minimal residual disease in a subset of patients in remission.

Electric field-mediated gene transfer into K562 cells: optimization of parameters affecting efficiency.

Since hemopoietic cells are refractory to transfection by conventional chemical means, we have developed a reliable and efficient gene transfer system for K562 cells which uses electric field-mediated gene transfer (EFMGT). EFMGT involves the exposure of cells in suspension to an electric field which transiently allows the entry of DNA into the cell and its subsequent integration and expression. Plasmids bearing the neo gene were used to identify and select transfected clonogenic cells manifested by geneticin resistance in semisolid medium. Transfection efficiency is significantly affected by the following variables: voltage, capacitance, time constant, number of pulses, buffer type and temperature, DNA concentration, configuration, and promoter type. Cell cycle status also appears to be critical as shown in studies employing aphidicolin synchronization. Using optimal conditions, we have consistently achieved a transfection efficiency of 0.3-0.4% of clonogenic cells per microgram DNA. Stability of neo gene expression was also demonstrated after 4 months in nonselective culture conditions. This level of efficiency compares favorably with other reports of gene transfer into human hemopoietic progenitor cells.

Long-term bone marrow culture induces terminal differentiation of human myeloid leukemic cells.

Bone marrow cells from a patient with acute myeloblastic leukemia were simultaneously cultured in vitro under conditions that favored the survival of either (1) leukemic progenitors (leukemic suspension culture), or (2) normal progenitors (long-term bone marrow culture). Whereas cells that were morphologically primitive and cytochemically leukemic persisted in leukemic suspension culture, they were progressively and completely replaced in long-term bone marrow culture by neutrophilic granulocytes and subsequently by macrophages. However, Auer rods were present in the maturing myeloid cells, including polymorphonuclear neutrophils, between the 7th and 30th days of long-term bone marrow culture, indicating that they were derived directly from the original leukemic population. This observation suggests that, at least in some patients, selection of cells with the potential for terminal differentiation may be the underlying mechanism responsible for the purging properties that have been attributed to long-term bone marrow culture.

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.

Characterization of K562 cells following introduction of a mutant N-ras gene.

A mutant human N-ras gene (codon 61, C to A substitution) was electroporated into the human leukemic cell line K562, originally derived from a patient with chronic myeloid leukemia (CML) in blast crisis. Despite confirmation of mutant N-ras gene integration and expression, mutant transfected cells exhibited no growth advantage when characterized in suspension cultures and clonogenic assays, and serum deprivation impaired proliferation of both normal and mutant N-ras transfected cells equally. A subclone containing a mutant N-ras gene displayed a proliferation rate and differentiation potential identical to that of non-transfected cells. The failure of N-ras mutations to modify K562 cell behavior is in keeping with the infrequent observation of N-ras mutations in blastic transformation of CML.

Thalidomide responsive chronic pulmonary GVHD.

A 29-year-old male underwent allogeneic bone marrow transplantation for progressive multiple myeloma. His post-transplant course was complicated by severe chronic pulmonary graft-versus-host disease (GVHD) resistant to cyclosporin A, corticosteroids and azathioprine. The introduction of thalidomide resulted in a dramatic improvement in his lung function which has been maintained even after cessation of thalidomide. He remains well 40 months after transplantation.

Rapid Screening of Mutant N-ras Alleles by Analysis of PCR-Induced Restriction Sites: Allele Specific Restriction Analysis (ASRA).

We have developed a rapid screening method for analysis of codon 12, 13 and 61 N-ras gene mutations, since these mutations have been observed in approximately 25% of patients with acute myeloid leukemia and myelodysplastic syndromes. The method, termed allele specific restriction analysis (ASRA), involves polymerase chain reaction amplification of DNA or RNA using a mismatched primer which introduces appropriately positioned base substitutions in N-ras and creates a restriction site provided the adjacent sequence is normal. Simultaneous analysis of codons 12 and 61 is also possible by the use of a multiprimer reaction mixture. Resistance of the amplified product to digestion indicates the presence of a mutation in the original template. Since ASRA allows simultaneous analysis of mutant and wild type sequences in DNA and RNA, an estimate of the ratio of gene copies and relative expression of N-ras alleles can be obtained for heterozygous individuals.

Disseminated granulocytic sarcoma treated with allogeneic peripheral blood stem-cell transplantation.

Primary granulocytic sarcoma is a rare disorder that presents as an extramedullary myeloid cell tumour. In this case, we describe a 35-year-old woman who developed widely disseminated granulocytic sarcomas three months after she received local radiotherapy to a primary granulocytic sarcoma of the cervical spine. These disseminated sarcomas initially responded to combination chemotherapy but this approach was only partly successful as the woman was left with increasing residual disease in the mediastinum. She was then treated with an allogeneic peripheral blood stem-cell transplant that led to complete resolution of her disease. This case demonstrates the effectiveness of allogeneic stem-cell transplantation in the treatment of progressive granulocytic sarcoma present after high-dose chemotherapy.

Late Relapses in Hodgkin's Disease: Are They a Distinct Entity?

It is claimed that late relapses of Hodgkin's disease have a good prognosis when retreated. A number of unfavourable outcomes led us to review our experience of 72 consecutive cases of Hodgkin's disease diagnosed and treated in a combined clinical haematology and radiation oncology unit between 1968 and 1984. 62 of 72 patients (86%) achieved a complete remission and of these, 35 patients (56%) relapsed, 18 occurring more than three years after diagnosis. Thus, 4 patient groups were identified: 10 patients with refractory disease, 27 patients who went into complete remission and have not relapsed, 17 patients who relapsed within 3 years of diagnosis (early relapse) and 18 patients who relapsed more than 3 years from diagnosis (late relapse). Patients who relapsed were retreated with well accepted protocols of chemotherapy and/or radiotherapy with surprisingly poor results. There was no significant difference between the survival from relapse of patients who relapsed early compared to those who relapsed late. Late relapses are not uncommon in Hodgkin's disease and the prognosis may be less favourable than generally perceived. The risk of relapse was almost constant with time and brings into question the concept of early and late relapse.

Overcoming methotrexate resistance by a lipophilic antifolate (BW 301U): from theory to models to practice.

We have provided a rationale for the clinical use of a new lipid-soluble folate antagonist, BW 301U, in terms of its potential for killing several classes of methotrexate-resistant cells. As part of a Phase I evaluation of this agent we studied normal bone marrow from cancer patients and their metabolic susceptibility to either BW 301U or to MTX and then repeated the observations at the end of five days of BW 301U infusions. Both inhibitors were roughly comparable at equimolar concentrations prior to therapy, but a relative resistance developed to MTX after BW 301U treatment. Such findings were replicated in an in vitro HL-60 cell culture system that was exposed to BW 301U. Some possible mechanisms for this unusual collateral resistance are discussed.

High-resolution analysis of gene rearrangements in lymphoid malignancies.

As T-cell receptor and immunoglobulin gene rearrangements provide specific clonal markers for lymphoid cell proliferations, analysis of these genes is useful for distinguishing between reactive and malignant disease. We have developed an automated, high-resolution analysis of PCR fragments to identify clonally rearranged TCR-gamma (TCR gamma) genes and IgH genes. Consensus primers are used to detect the majority of possible rearrangements in multiplex PCR assays, and the PCR products are fluorescently labelled for visualisation with ABI Genescan software. Polyclonal populations of lymphoid cells are represented by a spectrum of fragments, whereas a monoclonal population of cells is represented by one or two discrete bands, indicating rearrangement of one or both alleles. For TCR-gamma PCR, the rearranged DNA fragment from a monoclonal population of T-cells diluted to 0.1% in DNA from a polyclonal population of cells is still readily distinguishable from the polyclonal background. Similarly, for IgH PCR, the gene rearrangement from a monoclonal population of B-cells is still distinguishable to 0.5% in a polyclonal background. As this technique allows semi-quantitative resolution of fragments one base different in size, it is ideal for detecting monoclonal and oligoclonal populations of B- and T-cells. The accurate size determination of PCR fragments also minimises the risk of false positives resulting from contamination, as individual monoclonal rearrangements are frequently patient-specific on the basis of size alone.

Analysis of N-ras gene mutations in acute myeloid leukemia by allele specific restriction analysis.

N-ras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of N-ras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant N-ras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. N-ras mutations were more common in patients over 65 years of age (P less than 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of N-ras mutations in leukemia by ASRA and include the first identification of three concurrent N-ras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of N-ras mutations in leukemogenesis.

Induction of 2',5'-oligoadenylate synthetase activity in peripheral blood mononuclear cells by gamma interferon.

Activity of the interferon-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase was measured in peripheral blood mononuclear cells (PBMC) of 32 cancer patients treated with recombinant human gamma-interferon (rHGIF). Pretreatment activities varied widely but remained constant for each individual. The minimum dose of rHGIF that consistently stimulated activity was 12 MU/m2, although in some patients as little as 0.004 MU/m2 was stimulatory. Enzyme activity in responding patients increased threefold at 24 h after the initial infusion and remained elevated nearly twofold at 72 h independent of dose. This increase is less than that observed after infusions of alpha-interferon and parallels results observed in vitro. Pretreatment enzyme activity did not correlate with response. Twenty-minute infusions of a fixed dose were as effective as four- and 24-h infusions in raising enzyme levels. The level of activity achieved after the initial infusion could not be sustained with twice-weekly infusions. We conclude that rHGIF does induce 2-5A synthetase activity in PBMC but not to the levels induced by alpha-interferon. The observation that brief infusions of low doses can be maximally stimulatory in vivo suggests that the response of PBMC in vivo is being confounded by factors yet to be determined. Furthermore, it appears that the capacity of PBMC to maximally respond to rHGIF in vivo, with increases in enzyme activity, is transient.