Molecular Haemato-Oncology at Bristol Genetics Laboratory

1. Molecular Haemato-Oncology at Bristol Genetics Laboratory Kayleigh Templeman http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

2. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

3. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

4. Minimal Residual Disease (MRD) analysis • Used to monitor disease levels in children with ALL (acute lymphoblastic leukaemia). • Remission is defined as leukaemic cells being no longer detectable by light microscopy, but there could still be up to 5 x 1010 cells – this is the minimal residual disease. • MRD status following induction therapy is the single most important prognostic indicator in children with ALL. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

5. Patient’s diagnostic sample is screened for Immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements. This gene rearrangement is a normal process in the development of lymphocytes. • It can be assumed that these junctional regions are unique in each lymphoid precursor cell. • In theory, all of a patient’s leukaemic cells originated from a single clone, and therefore all the malignant cells should have identical Ig/TCR rearrangements.

6. Minimal Residual Disease (MRD) From the diagnosis sample we are aiming to identify 2 MRD markers that can be used to quantitate disease to a level of 1 leukaemic cell in 10,000 normal cells (10-4). Rearrangements identified are sequenced, and patient-specific primers created that can be used to detect disease in follow-up samples. Assays are carried out by real-time PCR, using a dilution series created from the patient’s diagnostic DNA, in order to quantitate any disease detected.

7. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

8. IgVH mutation testing in CLL Disease progression is very varied, so prognostic indicators are very important. IgVH mutational status in the leukaemic clone affects prognosis. - Mutations = 295 month median survival - No mutations = 95 month median survival (Somatic hypermutation ≥ 2% divergence from germline sequence) Clonal gene rearrangements are identifed and sequenced using a very similar method to the first stages of MRD analysis. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

9. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

10. Ig/TCR Clonality Assessment Requested when there is uncertainty as to whether a lymphoid mass is malignant. Test detects Ig/TCR gene rearrangements. A normal, not malignant (polyclonal) lymphoid cell population will contain the whole repertoire of gene rearrangements, and when analysed PCR products will give a polyclonal spread of peaks. A malignant cell population will be clonal, and produce a single peak. Patient http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

11. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

12. FLT-3 and NPM1 testing in AML Patient 1 An internal tandem duplication (ITD) in the FLT-3 gene is found in ~25% of adult AML and ~15% of childhood AML.  Poor prognosis Mutations in the NPM1 gene occur in ~35% of AML patients.  Good prognosis. Patient 2 Patient 3 Patient 4 http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

13. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

14. BCR-ABL1 Qualitative Analysis (RT-PCR) The BCR-ABL1 fusion gene is formed by a reciprocal translocation between chromosomes 9 (ABL1) and 22 (BCR)  Philadelphia (Ph) chromosome 90% of CML 20% of adult ALL 5% of childhood ALL 1% of childhood AML Patient Patient Patient +ve -ve 1 2 3 control control • 385bp (b3a2) http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

15. BCR-ABL1 Quantitative Analysis (RQ-PCR) Molecular monitoring of BCR-ABL1 is vital to the management of Ph +ve CML/ALL. Residual disease monitoring usually commences once a patient is in cytogenetic remission and allows for the assessment of response to treatment and identification of patients at risk of relapse. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

16. ABL1 kinase domain (AKD) mutation screening Recommended when a patient is not optimally responding to treatment, or when there is a loss of response to treatment so disease levels start to rise. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

17. Condition Test http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

18. JAK2 Val617Phe (V617F) mutation ~98% of patients with Polycythaemia Vera (PV) ~50% with Essential Thrombocythaemia (ET) or Idiopathic Myelofibrosis (IMF). Testing carried out by pyrosequencing. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

19. JAK2 Exon 12 mutation screen A small proportion of JAK2 V617F –ve patients have been shown to have mutations in exon 12. Mutations are detected by HRM and then characterised by direct sequencing. Difference Plot Normalised Melt Curve Patient http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

20. MPL mutation testing 3-4% of ET patients and 4-8% of IMF patients have mutations within Exon 10 of the MPL gene. MPL gene encodes thrombopoietin receptor. Patient – G T mutation at residue 1544 of codon 515 (most common mutation) TGG  TTG = tryptophan  leucine (MPLW515L) http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

21. Thank you for listening. Any questions? MRD team Dr Jeremy Hancock Service lead Paul Archer Lead Technician Alison Stevens Adiela Chudley Molecular Oncology team Dr Paula Waits (Mat Leave) Jennifer Corfield / Rebecca Wragg Kayleigh Templeman (kayleigh.mcdonagh@nbt.nhs.uk) http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory