Research Interest

Haemato-Oncology Molecular Diagnostic Service

Molecular diagnostics relies on a range of different technologies to identify changes genetic within the cancer cell.  The aim of detecting such abnormalities is to either predict a specific clinical outcome or to identify a tumour, which will respond to a specific therapy.  Molecular diagnostics, such as this, are essential to the development of stratified medicine approaches where a therapy is personalised to the genetic make up of a given patient’s tumour. This approach is essential to improve response rates, decrease side effects and to improve the value of expensive cancer chemotherapeutics.

The clinical relevance of molecular genetic analyses in haematological malignancies including the leukaemias and lymphomas is becoming increasingly well defined. Useful strategies involve the finding of mutations, differential gene expression profiles, translocations, deletions or amplifications and, more recently, epigenetic changes that can be used to characterise a particular disease state, helping in the differential diagnosis of similar entities or, within the same malignancy, genetic markers can help differentiating between subgroups with different clinical outcome. Disease-specific and/or patient-specific genetic markers can be also used to monitor minimal residual disease (MRD) by real-time PCR.

Molecular genetics in haemato-oncology are important to the point that the World Health Organisation (WHO) now includes now a variety of molecular features that are mandatory criteria for the differential diagnosis of certain types of Acute Leukaemia and Non-Hodgkin’s lymphomas. Also, studying such molecular abnormalities in tumour cells can tell us how patients with a given abnormality are going to respond to current treatments. For example, for the diagnosis of Acute promyelocytic leukaemias (or AML-M3) it is necessary to confirm the presence of the translocation t(15;17) which gives rise to the chimeric oncoprotein PML-RARA which, in turn, will lead to the clinician to treat such leukaemias with trans-retinoic acid (ATRA) and/or Arsenic trioxide, achieving a rate of complete remissions of >80%.  These clinical abnormalities and their therapies are some of the earliest examples of personalised medicine strategies in cancer therapy and the aim is to develop and apply more examples to this approach.

The information gained from the study of prognostic factors for particular diseases are relevant in the clinic, as they can give an idea of how a patient’s disease will behave. An example of this is the level of somatic hypermutation in the Ig genes of patients with Chronic Lymphocytic Leukaemia (CLL); CLL patients with high level of mutations in their Ig genes have a median survival of up to 25 years, whilst it drops down to 9 years for those CLL patients without mutations on the Ig genes.  Further more as described in CLL research section we do TP53 mutational status to identify patients with lower response rates and survival who might benefit from more personalised treatment and allogeneic transplants.

The Haemato-Oncology Molecular Diagnostics service is CPA accredited and provides fast and reliable genetic tests to help in the diagnosis and monitoring of patient’s disease. We have adopted the newest available technologies in order perform more reliable molecular tests, in less time, and in a more cost-effective fashion. For the study of chromosomal translocations in Acute leukaemias (AML and ALL), Real-Time quantitative PCR is performed for t(15;17), t(9;22), t(8;21), t(12;21), inv(16), t(1;19), t(4;11) and del(1), coupled with the study of the status of mutations of relevant genes that have an impact on diagnosis or prognostic, such as FLT3 in AML or JAK2 in MPD. We also perform clonality studies using PCR of the Ig receptors or the TCR genes for suspected chronic and acute malignant lymphoproliferations.  In the case of CLL, we routinely analyze the level of somatic hyper mutations of the Ig heavy chain genes. Patients undergoing allogeneic stem cell transplantation are routinely monitored by chimerism analysis, using a panel of 16 different fluorescent markers that provides a sensitivity <3%. Further mutation and translocations tests are also available, such as p53 and KIT mutations, as well as t(14;18).

Team Members
David Gonzalez, Senior Clinical Scientist
Lisa Thompson, Clinical Scientist
Dorte Wren, Clinical Scientist
Debbie Mair, Clinical Scientist
Carl Beyers, Biomedical Scientist
Chantal Hooper, Biomedical Scientist
Belen Gomez, Genetic Technologist
Jolyon Travis, Associate Practitioner
Dee Collins, Laboratory Administrator

Flow Cytometry and Immunophenotyping

In order to complement the molecular genetic characterisation of tumour cells, it is important to be able to study the protein markers expressed on their cell surface and intracellularly.  This can be done using staining with specific fluorescent antibodies combined with a detection system called a ‘flow cytometer’. In order to study intraclonal variation and the behaviour of leukaemia, at a single cell level, we have developed cell sorting technologies able to isolate single cells, which are amenable to further characterisation.

This is a specialised CPA accredited Haemato-Oncology Diagnostic Laboratory, which is able to provide an integrated diagnosis, based on the results of morphology, immunophenotyping, mutation and molecular genetics.  This approach allows us to definitively diagnose the leukaemias, myeloma and lymphomas. In addition to diagnostic tests we are able to provide tests to monitor the response of the disease to treatment. We carry out multiparameter flow cytometry for membrane and intracellular antigens using 4 colour techniques.  We process bone marrow, peripheral blood or lymph node specimens for:

• Immunophenotyping of acute leukaemia
• Chronic lymphoproliferative disorders
• CML
• Blast crisis  
• Immunophenotyping of myeloma and residual disease monitoring
• Minimal residual disease in acute and chronic leukaemias
• Prognostic markers for CLL, including CD38 and Zap 70
• Test for lymphocytes subsets for research or drug trials
• Stem cell enumeration (CD34) in peripheral blood, bone marrow and harvest

Team members
Saman Hewamana, Consultant Haematologist
Estella Matutes, Reader/Consultant Haematologist   
Mr Ricardo Morilla, Head of Immunophenotyping & Clinical Scientist
Mrs Alison Morilla, Laboratory Manager & Clinical Scientist
Mrs Katarzina Adamowska: Biomedical Scientist
Kelly Marquardt, Biomedical Scientist
Claire Stephens, Specialist Biomedical Scientist
Sophie Duke, Biomedical Scientist
Randeep Donapati, Biomedical Scientist
Athanasia Zeisig, PhD Biomedical Scientist
Christine Stuttle, Receptionist

Clinical Cytogenetics

Metaphase cytogenetics, looks at dividing cells synchronised in metaphase, and is a technique for visualising whole chromosomes and can, therefore, scan for changes across the whole genome.  Fluorescence Interphase Situ Hybridisation (FISH), is a cytogenetic technique able to detect abnormalities at a limited number of specific sites.  In a variation of this technique, interphase FISH (iFISH), it is possible to study cells that are not undergoing cell division i.e. they are not in metaphase.  This version of the technique can be used to study single cells and tissues, which have been processed making it a versatile readily applicable approach, but only when looking for a specific lesion.  While we have focussed activity on the development of molecular genetic testing able to identify predictive and prognostic biomarkers, there remains a number of lesions that can only be detected by cytogenetic strategies.  The laboratory provides a diagnostic cytogenetic service for patients with haematologic malignancy.  Including:

• Conventional metaphase cytogenetics and FISH
• Interphase FISH, (iFISH)
• Interphase FISH on Nuclei extracted from paraffin embedded material

Team members
Ms Carol Brooker, Consultant Clinical Cytogeneticist
Mr John Swansbury, Principal Cytogeneticist
Ms Alison McCormick, Principal Clinical Cytogeneticist
Mrs Shilani Aruliah, Senior Clinical Cytogeneticist
Ms Melissa Dainton, Clinical Scientist
Mrs Emma Fowler, Clinical Scientist
Dr Hend Hendow, Clinical Scientist
Dr Manisha Maurya, Clinical Scientist
Dr Toon Min, Senior Clinical Scientist
Dr Husam Salman, Clinical Cytogeneticist
Miss Tracy Stubbs, Clinical Scientist
Mrs Marianne Wall, Clinical Scientist
Miss Nasrin Dahir, Medical Laboratory Assistant