Molecular Cytogenetics Group
Group Leader: Dr Elisabeth Nacheva
Molecular Cytogenetics is concerned with the identification of genetics anomalies associated with the diagnosis and progression haematological malignancies. We have developed and extended the use of fluorescent in-situ hybridisation (FISH), array comparative hybridisation (aCGH) and next generation sequencing (NGS) for diagnostics. We are currently collaborating with Dr Panos Kottaridis seeking correlations between mutation status and response to therapy and Dr Christos Prokakis mapping variations in copy number changes in the brain of patients with Parkinson’s disease.
1. Next Generation Sequencing (NGS) in haematological malignancies. Every new generation of medical technologies forecast the demise of long established methods, so it is not surprising that ‘Cytogenetics is Dead’ (again). However, it is important to consider the benefits of very high throughput low cost sequencing that can, in principle at least, provide a diagnosis of all our ailments from a single DNA sample.
We have embarked on a program to evaluate the use of NGS for the detection of mutations recognised to have clinical significance in Leukaemia. Our NGS myeloid panel, for example, covers known myeloid genomic variant hotspots from 54 genes. All our data is held locally in digital format and is cross-referenced against patient history and treatments.
2. Molecular Karyotyping in Multiple Myeloma (MM) Screening for chromosome abnormalities, both numerical (hypo- and hyperdiploidy) and structural (IGH at 14q32 translocations, cryptic 13q14 deletions, and 1p loss/1q gain) present in 67-90% of the MM cases, is carried out by FISH on the bone marrow CD138(+) malignant B cells. This requires several (up to 8 ) separate tests. Now we use genome arrays to screen DNA from the CD138(+) cells and obtain a full genome profile at higher resolution. The illustration shows two cases where FISH would have missed aberrations indicative of disease. The loss of 12p (arrow in case b) indicates a poor prognosis:
3. Molecular Karyotyping in Chronic Lymphocytic Leukaemia (CLL) Specific genome aberrations are recognised prognostic factors in CLL. FISH-based genome risk classifications have been used in clinical decision making for over a decade. However molecular karyotyping is gaining acceptance as an alternative that overcomes the cost and skill intensive limitations of FISH and provides comprehensive whole genome scanning. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. We are extending these techniques to routine diagnostics.
4. Does Ph (+) AML Exist? That is, is it a different disease from CML myeloid blast crisis? We have been researching Ph (+) diseases for some time and one result is the resolution of this question. It is an important one as the treatment regime may alter depending on whether the disease is categorised as blast crisis CML or Ph (+) AML. Our recent publication in BJH goes some way to resolving this issue. Graphic illustrates the difference, showing a deletion in the IgH gene characteristic of lymphoid disease.
5. Philadelphia Positive ALL and the Lymphoid Blast Transformation of CML. Chronic myeloid leukaemia is a heterogenous disease evolving to both a myeloid and lymphoid crisis stage. The lymphoid form of CML blast crisis is normally differentiated from Acute Lymphoid Leukamia by reference to the evolution of the disease from its chronic form that may have endured for several years before transforming. Our research led us to study several cohorts of patient samples using very high resolution genome array mapping, which identified genomic loci associated with of previously unrecognised sub classes of ALL. Graphic illustrates a 5 mbase deleted region in chromosome 14 we have demonstrated is typical of B-cell disease:
Grace, C. & Nacheva, E. P. Significance Analysis of Microarrays (SAM) Offers Clues to Differences Between the Genomes of Adult Philadelphia Positive ALL and the Lymphoid Blast Transformation of CML. Cancer Inform 11, 173–183 (2012).
6. Resistance to Tyrosine Kinase Inhibitors. One of the great success stories in the battle against cancer is the development of a class of drugs known as Tyrosine Kinase Inhibitors (TKI). As seems to be the general case, leukamia patients in common with other cancer sufferers will develop resistance to the drugs. We have made a small contribution to identifying the mechanism by which the resistance is acquired.
• Dr Diana Brazma
• Julie Howard-Reeves
• Dr Nushka Bonneva
• Hashim Bakhshi
• Katya Gancheva
• Prof S Mackinnon – Dept. of Haematology, UCL
• Dr P Kottaridis – Dept. of Haematology, UCL
• Prof V Najfeld - Mount Sinai Hospital, NJ, USA
• Dr C Prokakis – Dept. of Neurology, UCL