Kenneth Ian Mills

c-myc locus amplification and the acquisition of trisomy 8 in the evolution of chronic myeloid leukaemia

The biological progression of chronic myeloid leukaemia is often associated with secondary cytogenetic abnormalities but the molecular mechanisms underlying this progression are poorly understood. This study explores the association of c-myc gene amplification with the progression of chronic myeloid leukaemia in fourteen individuals. Three of these cases showed amplification of c-myc during the course of their disease. Cytogenetic and molecular analysis of serial samples from some patients suggested the successive expansion of distinct clones of malignant cells. Our findings also suggest that trisomy 8 and locus amplification could represent alternative mechanisms for increasing c-myc gene dosage.

Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia.

Myelodysplastic syndromes (MDS) are characterized by a clonal disorder of haemopoiesis with defective growth in vitro. The long‐term culture system was used to examine aspects of stromal function in MDS patients. Primary long‐term cultures of MDS bone marrow showed poor myelopoiesis with progenitors being detected for a median 3·5u2003weeks (nu2003=u200312) compared with 18u2003weeks in cultures of normal marrow (nu2003=u200310; Pu2003<u20030·0001). The haemopoietic function of adherent layers was assessed in secondary co‐cultures seeded with 5u2003×u2003106 cord blood mononuclear cells on irradiated normal (nu2003=u200327; aged 38–82u2003years) or MDS (nu2003=u200332; aged 41–86u2003years) adherent layers (>u200360% confluent). The median myeloid progenitor number/cord blood co‐culture was 135 in 5‐week‐old cultures with normal adherent layers and 22 in those with MDS layers (Pu2003<u20030·0001). Myeloid colonies were detectable for a median 11u2003weeks with normal adherent layers and 6u2003weeks with MDS adherent layers (Pu2003<u20030·0001); erythroid colonies were detectable for 7u2003weeks (normal) compared with 5u2003weeks (MDS) (Pu2003<u20030·01). The differences in granulocyte‐macrophage colony forming unit (CFU‐GM) generation were not related to patient age. Cells from adherent layers of at least half of the primary normal (nu2003=u200348) and MDS (nu2003=u200326) long‐term cultures expressed cytokines [interleukin (IL)‐3, IL‐1β, thrombopoietin (Tpo) and erythropoietin (Epo)] and receptors for retinoic acid (RARα) [IL‐2, IL‐3, macrophage colony stimulating factor (M‐CSF) (Fms) and Tpo (Mpl)]. Only IL‐1β expression was reduced in week‐5 MDS cultures compared with those from normal marrows (Pu2003<u20030·05). There was also a highly significant decline in IL‐1β expression in normal (but not MDS) adherent layers between weeku20035 and weeku200310. Thus, the adherent layers in cultures grown from MDS patients were haemopoietically defective and showed abnormal IL‐1β expression.

p53 Mutations, Methylation and Genomic Instability in the Progression of Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), as with other tumour types, mutations of the p53 gene are associated with disease progression. Changes in regional methylation of DNA with CML tumour development have also been demonstrated. Methylation is one mechanism by which gene expression is controlled and the CpG sites, which are the targets of DNA methylation, are also the sites of a number of the mutations found in the p53 gene. Cells harbouring mutant p53 have been shown to accumulate further genomic and genetic aberrations and methylation which alters the conformation of DNA is also believed to play a role in genomic stability. There appears to be an interplay between p53 deregulation and changing methylation patterns with the progression of CML. The cause and effect of changes in both of these critical gene regulating, DNA repair and genomic stability factors and their deviation during the progression of CML will be discussed.

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all‐trans‐retinoic acid (ATRA) and 1α,25‐dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to ‘anti‐inflammatory agents and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up‐regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3.

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA‐500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

Homozygous MHC Genotypes and Longevity

To investigate the relevance of the major histocompatibility complex (MHC) in longevity, we carried out a molecular analysis of the MHC in 432 unrelated healthy individuals. The comparison of individuals < or = 25 years and > 25 years showed that the 5.8-kb DQA1 allele, which corresponds to HLA-DR53, was negatively associated with longevity (p = 0.0035) resulting mainly from decreased homozygosity with age for that allele (p = 0.008), and restricted to males (p = 0.008). The difference was more striking for the 5.8 kb DQA1: 9.0 kb HSP70 haplotype again only in males (26.3 vs. 6.2%; p = 0.017, OR = 5.4, 95% CI = 1.5 - 19.5). The oldest male subject homozygous for this DQA1: HSP70 haplotype was 54 years (p = 0.005). Comparing leukemic patients and healthy individuals with the same ethnic and geographical origin, homozygosity for these genotypes was more frequent in the young leukemic group. The results suggested the existence of recessive deleterious genes in a segment of HLA-DR53 haplotypes.

High FUS/TLS expression in acute myeloid leukaemia samples

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all‐trans retinoic acid (ATRA) for only 1u2003h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1u2003h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto‐oncogene. Northern analysis showed that FUS was downregulated within 1u2003h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA‐resistant cell lines did not show a downregulation of FUS over a 24u2003h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA‐sensitive and ‐resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

Changing p53 mutations with the evolution of chronic myeloid leukaemia from the chronic phase to blast crisis

The frequent involvement of chromosome 17p abnormalities in the progression of chronic myeloid leukaemia (CML) led us to investigate the involvement of the p53 tumour suppressor gene located on chromosome 17p. We analysed 31 samples from four patients sequentially, and 16 patients in blast crisis only, using single stranded conformational polymorphism (SSCP) analysis of exons 5-8, followed by cloning and sequencing. The sequential samples ranged from diagnosis through to late disease. We found that 15% of our blast crisis samples had p53 abnormalities. In our sequential studies we found two of the four patients analysed in more detail had p53 mutations in the late chronic phase of disease (11 and 5 months prior to blast crisis becoming apparent). These chronic phase mutations differed from the p53 abnormalities found in the blast crisis samples from these patients. One patient also had the same chronic phase mutation at post bone marrow transplant relapse. Our results suggest that, in some cases, sequential investigations through CML disease progression of p53 mutations and other oncogenes/proto-oncogenes may provide early indications of the routes of disease progression to blast crisis.

A simple PCR/RFLP analysis can differentiate between Candida albicans, Aspergillus niger, and Aspergillus fumigatus.

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.

An in vivo and in vitro comparison of the effects of b2-a2 and b3-a2 p210BCR-ABL splice variants on murine 32D cells.

The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CMJL), is caused by a reciprocal translocation juxtaposing the 3′ region of the ABL gene onto the 5′ region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal ewolution and cytogenetic instability in these cells alone. At 106 and 107 cell doses all 32D cells; expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 105 and 104 dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorten-survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferned by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This punel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.