R. A. Padua

Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.

Clonal lymphocytes are detectable in only some cases of MDS

Clonal analysis of lymphocytes from patients with myelodysplastic syndrome (MDS) has been carried out using X‐chromosome inactivation patterns detected by the probe M27β, and by polymerase chain reaction amplification of the immunoglobulin heavy chain gene hypervariable region, CDR3. Of 32 female patients heterozygous for M27β only seven (22%) demonstrate monoclonality of peripheral blood lymphocytes. 12 (37%) give unequivocal polyclonal results and the remaining cases give patterns of X‐inactivation which cannot be interpreted either way. A study of 68 MDS patients showed five (7%) with a population of B‐cells with a monoclonal rearrangement of CDR3 compared with none out of 60 normal individuals, none out of 15 with B‐non Hodgkin lymphoma (B‐NHL) in remission and 19 out of 25 (75%) of cases of B‐chronic lymphocytic leukaemia (B‐CLL). Monoclonal lymphocytes were found by both techniques in only two females with MDS. We conclude that the presence of polyclonal lymphocytes is a common finding in patients with MDS.

Frequent expression of HAGE in presentation chronic myeloid leukaemias

Cancer testis (CT) antigens provide attractive targets for cancer-specific immunotherapy. Although CT genes are expressed in some normal tissues, such as the testis and in some cases placenta, these immunologically protected sites lack MHC I expression and as such, do not present ‘self’ antigens to T cells. To date, CT genes have been shown to be expressed in a range of solid tumours, but rarely in haematological malignancies. We have extended previous studies to investigate the expression of a comprehensive range of CT genes (MAGE-A1, -A3, -A6, -A12, BAGE, GAGE, HAGE,LAGE-1, NY-ESO-1 and RAGE) for their expression in a cohort of acute and chronic myeloid leukaemia patient samples. CT expression was not detected in 20 normal bone marrow or peripheral blood stem cell samples. In acute myeloid leukaemia (AML) nine of the 26 (35%) samples analysed expressed one or more of the CT genes with six of the samples (23%) expressing HAGE. In chronic myeloid leukaemia (CML) 24 of 42 (57%) presentation chronic myeloid leukaemia (CML) patient samples expressed one or more CT antigen with 23 expressing HAGE. We have shown that HAGE is frequently expressed in CML, and to a lesser extent in AML patient samples. This is the first demonstration of HAGE gene expression in myeloid leukaemia patients and the frequent expression of HAGE at disease presentation opens up the possibility of early immunotherapeutic treatments.

The application of X-chromosome gene probes to the diagnosis of myeloproliferative disease.

Summary X‐chromosome DNA probes for the phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl transferase (HPRT) genes were used to study clonality in haemopoietic cells from 63 women with myeloproliferative disease, idiopathic erythrocytosis. secondary erythrocytosis or normal red cell volumes. A total of 25 women (39%) were heterozygous for one of the polymorphisms associated with these genes. Clonality was demonstrated in five out of six patients with polycythaemia vera (PV) and in three other patients with myeloproliferative disease. In all cases of PV, including the patient in whom clonality was not demonstrated, cultures of peripheral blood showed growth of endogenous erythroid colonies.

Oncogene mutation and prognosis in the myelodysplastic syndromes.

In a series of myelodysplastic syndrome patients, mutational status (particularly RAS mutation) was found to be prognostic for survival, independently of four previously reported scoring systems (Bournemouth, Sanz, Lille, International).

Chromosomal assignment of c-MEL, a human transforming oncogene, to chromosome 19 (p13.2-q13.2).

The human malignant melanoma cell line NKI4 contains a novel transforming gene which was identified using DNA transfection into NIH3T3 cells (1). This gene has been assigned to chromosome 19p13.2-q13.2 using human-mouse and human-hamster somatic cell hybrids.

ras Mutations in the Myelodysplastic Syndromes

The ras gene family—H-ras, K-ras and N-ras— code for 21-kDa proteins that have GTPase activity and have been implicated in the control of cell proliferation [8,13]. Mutations in these genes give rise to abnormal protein products that have the capacity to transform certain cells to a malignant phenotype. The ras mutations have been found in a wide range of human malignancies, and N-ras has been particularly implicated in acute myeloid leukaemia (AML), chronic myeloid leukaemia and acute lymphoblastic leukaemia [4, 5]. Activation of these genes has been associated with mutations in codons 12/13 or codon 61, and lesions of this type have recently been described in myelodysplastic syndromes (MDS). Hirai et al. [3] described three MDS patients with codon 13 mutations in N-ras,and Liu et al. [6] reported two patients with a similar mutation in K-ras. We have assessed the mutational status of members of the ras gene family by polymerase chain reaction and hybridisation with synthetic oligonucleotide probes in 50 patients with MDS together with the use of a nude mouse tumorgenicity assay in some cases [9].

Screening for retinoic acid receptor alpha (RAR alpha) rearrangements in non-APL identifies a novel fusion gene in CMML

The mechanisms underlying the commitment of a hematopoietic stem cell to an individual lineage are largely unknown. To gain insight into the molecular events associated with the commitment of !hese totipotent cells to the megakaryocylic lineage, a strategy was developed to generate mice in which a reversible cell knock out at a specific stage of the differentiation process could be induced. This was accomplished both, by introducing the gene encoding the herpes simplex virus thymidine kinase into the integrin (Lllb locus. using homologous recombination technology and by additional transgenesis. This approach provided a convenient manner to induce the programmed eradication of cells expressing the conditional toxigene by treatment with the antiherpetic drug ganciclovir, and resulted in the parallel knock out of the endogenous q l b gene. Platelets collected from these ctllb deficient mice failed to bind fibrinogen and to aggregate. Clot retraction was also impaired and mice were unable to control blood loss following surgical challenge. Consistent with these characteristics these mice displayed bleeding disorders similar to the human Glanzmanns thrombasthenia pathology. Results of progenitor cell assays combined with long term bone marrow cultures performed on slromal layers revealed that the allb glycoprotein is dispensable for lineage commitment, megakaryocytic maturation and established that the toxigene is expressed in a subset of totipotent stem cells. This demonstrates that genetic activation of lineage specific genes occurs prior to the commitment. strongly supporting the concept that stem cells express a multilineage transcriptome. -1TRlLlNEAGE RESPONSES SEEN WITH STEM CELL FACTOR (STERRGEW, SCF) AND FlLGRASTlM (G-CSF) TREATfflENT IN APLASTIC ANEMIA (AA) PATIENTS (Pts)

A novel CSF-1 binding factor in a patient in complete remission following cytotoxic therapy for lymphoma.

Summary. A novel colony stimulating factor‐1 (CSF‐1) binding factor present in the serum from a patient in remission from lymphoma is described. Radioimmunoassay (RIA) repeatedly failed to detect circulating levels of CSF‐1 in the peripheral blood system of this patient. Molecular analysis showed a normal CSF‐1 gene structure by Southern blot analysis and a 46, XX karyotype by cytogenetic analysis. CSF‐1 mRNA expression in peripheral blood leucocytes was confirmed using reverse transcriptase polymerase chain reaction analysis. Morphological analysis of bone marrow cells was normal and peripheral blood progenitor cell colony assays showed a pattern of growth within the normal range in response to CSF‐1 alone and in combination with other cytokines. Analysis of the patients plasma and conditioned media prepared from peripheral blood mononuclear and granulocytic cell fractions for their ability to bind 125Iodine‐labelled CSF‐1 revealed the presence of a plasma CSF‐1 binding factor. This binding factor was not present in the patients urine, because CSF‐1 was detected by RIA and production of the binding factor by the patients peripheral blood white cells could not be demonstrated in vitro. To our knowledge, this is the first reported case of a soluble CSF‐1 binding factor.

ras Mutations in Preleukaemia, in Patients Following Cytotoxic Therapy and in Normal Subjects

Point mutations in members of the RAS gene family, NRAS, KRAS and HRAS, are a common molecular lesion in patients with acute myeloid leukaemia (AML). Approximately 30% of such patients show mutations (Bos, 1987; Farr et al., 1988) mostly in NRAS, although KRAS abnormalities are also seen. Mutations in HRAS are a rare abnormality in haemopoietic malignancies (Browett et al., 1989). A similar pattern of mutations is also seen in patients with the preleukaemic Myelodysplastic syndromes (MDS), where mutations have been detected in up to 41% of patients (Padua et al., 1988; Yunis et al., 1989). In MDS RAS mutation appears to be associated with poor prognosis in terms of an increased likelihood of transformation to acute leukaemia, and whilst RAS mutation can be detected in all subtypes of MDS (Padua et al., 1988), the highest incidence is seen those patients a monocytic phenotype (Padua et al., 1988; Yunis et al., 1989).