Research Programme

We are looking for DNA sequences that appear to be significantly associated with the disease stage and lineage origin in CML and Ph (+) ALL samples. Using array CGH data for 125 samples (92 CML, 22 Ph+ ALL and 11 controls) we have shown how Significance Analysis of Microarrays (Tusher et al Proc Natl Acad Sci U S A. 2001 April 24; 98(9): 5116–5121) can be used to discriminate between the genomes of lymphoid and myeloid blast cells and how consistent features emerge during the transition from the chronic to blast phases of the disease. Having identified regions of potential interest, ranked in order of significance, out of the 100’s of thousands of array results, we are facing the challenge of designing further experiments to evaluate their functions.

We identified an unexpected loss of the ABL1 (wild-type) sequences in the normal allele on the non-translocated homologue of chromosome 9 in three BCR/ABL1 positive CML patients on tyrosine kinase inhibitors (TKI) who developed therapy resistance. This loss was not present at the time of diagnosis but developed during disease progression and was found along with other additional chromosome rearrangements in the BCR/ABL1 positive cells. We studied bone marrow samples by array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analysis using a range of commercial and bacterial artificial chromosomes (BAC) probes. Sensitivity to TKI might be affected in the BCR/ABL1 positive cell clone bearing the deletion since it is claimed (ref) that negative control is exercised by the normal ABL1 allele over the BCR/ABL1.

We have studied BCR and ABL1 flanking regions and associated chromosomal rearrangements in 9 Ph negative BCR/ABL1 positive CML patients and the cell line CML-T1. The BCR/ABL1 fusion was located at chromosome band 9q34 in 3 patients, 22q11 in 5 patients (and the cell line) and 22p11 in 1 patient. In 3 of 6 cases with the fusion at 22q11, a distal breakpoint cluster was found within a 280 Kb region containing the RAPGEF1 gene, while in another patient and the cell line the distal breakpoint fell within a single BAC clone containing the 3' RXRA gene. Two cases had a duplication of the masked Ph while genomic deletions of the flanking regions were identified in 3 cases. Even more complex rearrangements were found in 3 further cases. We concluded that BCR/ABL1 fusion resulted from a direct insertion (one step mechanism) in 6 patients and the cell line, as opposed to a multistep mechanism in the other 3. The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. Future studies are being performed to confirm the presence of true breakpoint hot spots and assess their implications in Ph negative BCR/ABL1 positive CML.

Chromosome 17 is known to contain many genes and aberrations and this chromosome has been shown to be of particular interest in all fields of medical genetics, both acquired disease and constitutional disorders. In haematological malignancy, 17p11-13 deletions have been described in CLL, balanced translocations involving 17q22 are observed in APML and i(17q) is significant in CML. In constitutional syndromes, regions of Low Copy Repeat (LCR) sequences on chromosome 17 can lead to DNA rearrangements and human disease traits (Lupski et al) such as Miller Dicker Syndrome, Smith Magenis Syndrome, Charcot-Marie Tooth disease and the Hereditary Neuropathy to Pressure Palsy (HNPP), not to forget the cancer prone disorders, Neurofibomatosis1 (17q11.2) and even solid tumours.

The 17p11-13 is a region of the genome that is of particular interest in the field of haematological malignancy. This region contains the gene TP53 which is reported to be deleted in CLL and a recently described dic(17;18)(p11;p11) as a recurring aberration in CLL with aggressive disease (Woyach et al, 2009). We are undertaking a comprehensive high resolution aCGH survey of patients with 17p aberrations across a range of haematological diseases to identify common features and establish correlations with clinical data.

A 51 year-old woman diagnosed in 2007 with Philadelpia chromosome (Ph) and BCR-ABL1 positive CML achieved complete cytogenetic and molecular remission after treatment with Tyrosine Kinase Inhibitors (TKI) Imatinib Mesylate and Dasatinib. In July 2009 the patient presented with AML M1, associated with a complex karyotype. Analysis revealed balanced translocation t(2;3) disrupting the EVI1 gene at 3q26 as initial event followed by clonal evolution including monosomy 7, deletion of part of the long arm of chromosome 6 and t(4;8)(q12;q24). The presence of BCR/ABL1 fusion was ruled out by FISH and qPCR.

Clonal chromosome changes in Ph-negative cells are known to develop in some 10% of Ph(+) patients during TKI therapy. These abnormalities, mostly whole chromosome aberrations such as extra 8 or loss of 7, are transient and appear to have of no clinical consequence. Never the less, development of high risk MDS and AML have been reported during TKI treatment. Our findings demonstrate the involvement of EVI1 gene as initial event presiding the loss of chromosome 7 in Ph-negative cells of a CML patient on TKI treatment. Rearrangements of EVI gene have been associated not only with t(2;3) but also with a subtle chromosome abnormality inv(3)(q21q26), known to occur in blast transformation of CML as well as AML. Therefore the disruption of the EVI should be first line candidate for screening of TKI treated patients with clonal aberration in the Ph (-) bone marrow cells.

Array CGH profile of chromosome 9 shows increased copy number (amplification) of sequences from the 9q34.12 region in CML blast crisis.

FISH mapping confirms the aCGH results (a) and locates the extra copies within an apparently ‘normal’ Ph chromosome (b & c) ; for more details see poster “Double Ph” presented the ESH CML conference in Boston, USA, 5-7th Sept 2008.

AGH profile of chromosomes 7 & 9 showing loss of copy number (deletion). Smoothed data from very high resolution aCGH ~ 250bp (blue) confirmed the low resolution oligo results (red) in agreement with FISH mapping (data not shown); for more details see poster “Concomitant Loss” presented at the ESH CML conference in Boston,USA, 5-7th Sept 2008.

Numerous factors are uniquely associated with the lymphoid form of the disease for example the deletion of the Ikaros gene as illustrated here.

Genetic variations such as these shed light on the processes underlying disease onset and development.

Granulocyte colony stimulating factor (G-CSF) is a cytokine widely used for the collection of peripheral blood stem cells (PBSC) for autologous, sibling and unrelated haematopoietic stem cell (HSC) transplantation. A 4 days course of G-CSF is administered to stimulate the release of HSC from the bone marrow into the peripheral blood, in a process called mobilization.

While the short-term side effects of G-CSF are well known, mild and transient, there are concerns about the long-term safety and a possible role in leukemogenesis. Retrospective surveys in the US and Europe have not found increased levels of malignancies in mobilized healthy PBSC donors that have been followed for many years. However, a study by Nagler et al. (Exp Hematol, 2004) in a small cohort of G-CSF mobilized donors showed a temporary presence of altered gene replication timing and chromosome aneuploidies in their lymphocytes.

We are currently participating in a joint study of the Anthony Nolan Trust and the British Bone Marrow Registry on the impact of stem cell donation either after mobilization with G-CSF or bone marrow harvest on unrelated healthy donors. We are using FISH with a range of probes and array CGH to screen PBSC donors and BM donors for long-term genetic damage.

Human Herpesvirus 6 (HHV-6) has the unique ability to integrate into the genome of infected individuals. Those with this rare form of viral latency show persistent abnormally high levels of HHV-6 DNA without active infection. We have shown by FISH that the HHV6 integration occurs preferentially in the telomeric region of the chromosomes (Nacheva et al, 2008).

Furthermore, we have demonstrated that the integrated HHV-6 can also be transmitted through haematopoietic stem cell transplantation from the donor to the recipient as well as from parent to child, leading to high levels of DNA that may be misinterpreted as an active infection and prompt an inadequate toxic antiviral therapy.

We are using an HHV-6 specific FISH probe to confirm the genome integration and identify the chromosomal location in individuals with high viral loads.