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

Condition Test ALL CLL AML CML MPN Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing BGL is part of Bristol Haemato-Oncology Diagnostic Service (BHODs). Have access to a full range of complementary pathology services. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

Condition Test CLL AML CML MPN ALL Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 3

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. ALL = most common paediatric cancer Around 1012 leukaemic cells are present at diagnosis, making ALL easily detected with light microscopy. Normal BMA contains up to 5% of cells that are indistinguishable from leukaemic blasts. Remission is defined as leukaemic cells being no longer detectable by light microscopy. Therefore, at remission 5% of cells visible may still be leukaemic, meaning that the disease load could still be as high as 5 x 1010 cells – this is the minimal residual disease. UKALL 2003 trial finished earlier this year. UKALL 2011 interim trial in progress R3 (for patients who have relapsed) and Interfant 06 (for patients diagnosed <1yr old) trials in progress. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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.

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. MRD – It’s a long, expensive process! A month from diagnosis to Day 28, and you need every day! Marrow sample processing - density gradient separation of mononuclear cells DNA extraction Clonal rearrangements at immunoglobulin (Ig) and T-cell receptor (TCR) loci are detected using BIOMED screening PCR panels (made at BGL). PCR products run on acrylamide gels Heteroduplex resolution Sequencing Junctional analysis of sequencing products to identify unique junctional rearrangements. ASO primer design and testing using dilution series created from patient’s diagnostic DNA Real-time PCR analysis of patient samples at various timepoints throughout treatment

Condition Test ALL CLL AML CML MPN Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 7

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. TAT 20 days. PB or BMA. Analysis of Mutational Status of Clonal IGVH Gene Rearrangements in Chronic Lymphocytic Leukaemia CLL is the most common leukaemia in the Western world. Clinical progression of the disease is very varied, many survive for long periods without any particular therapy, but some have a much more rapid disease progression which requires immediate treatment, and many of these die despite aggressive therapy. Therefore, very important to be able to identify which patients are likely to go down this route, and so need prognostic indicators. One of these is the mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene segments in the patient’s leukaemic cells. Patients with these mutations have a median survival of 295 months, compared to 95 months for those with no mutations. However, patients with gene rearrangements involving the VH3-21 gene region have a poor outcome irrespective of mutational status. Clonal gene rearrangements are identified in a similar way as we do for the MRD assay, by PCR, gel and heteroduplex analysis. Clonal rearrangements are then sequenced , and V, D and J gene usage is determined by comparison to germline sequence databases (IMGT or IgBlast) and hypermutation analysis is undertaken to determine the divergence from the germline VH region. (Somatic hypermutation is defined as greater than or equal to 2% divergence from the germline variable gene segments) http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

Condition Test ALL CLL AML CML MPN Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 9

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 TAT 10 days Malignant lymphocytes accumulate in lymph nodes Usually diagnosis is performed using histomorphology, immunochemistry and flow cytometry. Sometimes though distinguishing between reactive lymphoproliferations (ie in response to an infection/inflammation) and malignant lymphomas is difficult. This is when immunoglobulin (Ig) and/or T cell receptor (TCR) clonality assessment can be useful As we have seen, Ig/.TCR rearrangements are a normal part of lymphocyte maturation, which each lymphocyte having its own unique rearrangements. Lymphoma is a clonal disease, all cells originated from one cell, so all will have the same rearrangement. Giving a single peak. They could contain one or several Ig/TCR rearrangements. Tissue containing normal lymphocytes, PCR will amplify the whole repertoire of gene rearrangements. Giving a polyclonal spread. DNA usually extracted from PETs, but also fresh/frozen tissue/tumours, PB, BMA. DNA quality tested using a basic PCR to create a DNA ladder, shows how fragmented DNA is Then carry out PCR - BIOMED-2 IdentiClone™ multiplex PCR systems – divided into initial (91%) and extended (100%) B screen (multiplex PCR panel), and initial (94%) and extended (100%) T. (detection rates for proliferations) B-screen for clonal rearangements - loci – IgH (Immunoglobulin heavy chain), IgK (immunoglobulin light chain kappa), IgL (immunoglobulin light chain lambda) T-screen loci – TCR G (gamma) , TCR B (beta), TCR D (delta) PCR products are then resolved using differential flourescence detection by capillary electrophoresis (Beckman Coulter CEQ8000) Then analysed, each locus has its own size range within which a valid clonal peak will appear. If peak appears within polyclonal background, it must be 3x higher than the 3rd highest peak in the polyclonal background. Results must then be used in conjunction with all other clinical, histological and immunophenotypic data information. A clonal peak can indicate a haematological malignancy, but it DOES NOT diagnose it. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 10

Condition Test ALL CLL AML CML MPN Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 11

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 Diagnostic/prognostic screen. 3-5ml blood in EDTA. 10 days TAT. Duplex PCR. Analysed on Beckman Coulter CEQ8000 and standard agarose gel electrophoresis to identify larger ITDs. Acquired molecular markers that give prognostic information. FLT-3 = fms-like tyrosine kinase-3 13q12.2. 24 Exons. FLT-3 itd is an in-frame duplication and tandem insertion of between 3 and 400bp in the juxtamembrane domain-coding sequence. This itd causes a constitutively activated receptor (tyrosine kinase activity). Poor prognosis – increased relapse risk, reduced survival (DFS, EFS and OS) NPM1 = nucleophosmin member 1 5q35. 12 exons. Most mutations occur in exon 12, ~40 identified so far. Mutation is nearly always a 4bp insertion caused by the duplication of 4 bases at position c.956-959. The most common (75-80% of cases) is TCTG, known as Type A. Type B = CATG (10%). Type D = CCTG (5%) NPM1 mutations are associated with a good prognosis – higher rate of complete remission (CR), longer EFS and stronger trend to OS than WT NPM1. Patients can be stratified into prognostic groups, with those in middle group either having both mutations or neither. These groups identified by the recent MRC AML 10/12 trial (Gale et al., 2008). Patient 4 http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

Condition Test CLL AML CML MPN ALL Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 13

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) Diagnostic screen. TAT 3 days for ?CML or 10 days for more general diagnostic enquiries. Lab also carries out cytogenetic and FISH analysis. BMA or PB in EDTA, must reach lab within 72 hours. Samples must be processed in clean DNA, Rnase free environment to remove red blood cells and produce aliquots of white cell lysate. RNA then extracted from lysates on Qiagen Qiacube. RT-PCR carried out to create cDNA. Tetraprimer PCR carried out using the cDNA, and the products run on an agarose gel in order to detect the major breakpoint transcripts (b2a2 (310bp) and b3a2 (385bp), most common) and the minor breakpoint transcript (e1a2(481bp)) Most BCR-ABL rearrangements occur within the major breakpoint cluster region. Hard pushed to get a result out in 3 days! Diagnostic, but if CML has been diagnosed then can be used to identify the transcript involved and determine whether the patient is able to be monitored by RQ-PCR (Real time quantitative PCR). http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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. TAT 10 days. 5-10 ml PB in EDTA, to reach lab within 72 hours for efficient RNA extraction. Same processing as previously, white cell isolation and lysis, RNA extraction, RT-PCR to produce cDNA, RQ-PCR. RQ-PCR using the Europe Against Cancer probes and primers outlined in Gabert et al., 2003. Control gene RQ-PCR allows for accurate quantitation of amplifiable cDNA. ABL gene used as control gene. BCR-ABL RQ-PCR then carried out, and BCR-ABL levels can be related to ABL levels to give a BCR-ABL:ABL ratio and allows for comparisons of BCR-ABL levels throughout treatment. Disease then defined as: Continuing/residual/molecular residual disease. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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. TAT 20 days. PB sample. BCR-ABL +ve CML/ALL is treated with tyrosine kinase inhibitors (original = Glivec/Imatinib) which block the ATP-binding site of the fusion protein. Mutations in the ABL kinase domain can prevent the drug from binding but still allow the binding of ATP. Standard RNA sample processing. Semi-nested PCR, followed by checker gel to ensure PCR has worked. PCR products then sequenced and sequence analysed for mutations. Depending on the mutation identified (or if a mutation is identified), patients could be offered 2nd generation TKIs (Dasatinib, Nilotonib), or maybe entered onto trials for new TKIs if it has been shown that their mutation will cause resistance to a particular/all TKI. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

Condition Test ALL CLL AML CML MPN Childhood Ph+ve Minimal Residual Disease (MRD) analysis Ph+ve BCR-ABL1 analysis (Quantitative and qualitative) ABL1 kinase domain (AKD) mutation screening CLL IgVH mutation testing Suspect lymphoproliferations Ig/TCR clonality assessment AML FLT-3 and NPM1 mutation testing CML ABL1 kinase domain mutation (AKD) screening MPN JAK2 and MPL mutation testing http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory 17

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. Diagnostic test. 10 days TAT. 3-5ml PB Detection of mutation allows distinction of true Myeloproliferative Neoplasms (MPN) from reactive disorder. Most MPNs (exclude CML) have no chromosome abnormality. JAK2 found at 9p24. GT point mutation in exon 14 of JAK2 results in valine to phenyalanine substitution at protein position 617. Results in deregulated tyrosine kinase activity. Pyrosequencing is a very rapid and quantitative method for detecting SNPs. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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 Diagnostic test. 20 day TAT. 3-5ml blood in EDTA, or DNA in storage from JAK2 test. Mutations in exon 12 again result in deregulated tyrosine kinase activity. Test is only performed if patient has been tested for JAK2 V617F. Patient http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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) PB in EDTA or stored DNA. TAT 20 days. MPL (Myeloproliferative leukemia virus oncogene) gene is on 1p34 Thrombopoietin regulates production of platelets and megakaryocyte development and proliferation Binding to receptor activates a number pathways including the JAK family of tyrosine kinases 6 mutations identified, 5 involving MPLW515 residue (tryptophan) (MPLW515L, MPLW515Ki, MPLW515Kii (lysine), MPLW515R, MPLW515A), other is MPLS505N (serine  asparagine) W515 residue is important for maintaining the receptor in an inactive state when thrombopoietin isn’t bound, this suggests that mutations in this residue cause the receptor to be constitutively active without the binding of thrombopoietin Mutations within exon 10 are identified by HRM, and confirmed/characterised by direct sequencing. http://www.nbt.nhs.uk/gps/services__referral/b/bristol_genetics_laboratory

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