32nd UK Cancer Cytogenetics Group Meeting 15 th & 16 th March Newcastle

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1 32nd UK Cancer Cytogenetics Group Meeting 15 th & 16 th March Newcastle The Director of the Leukaemia Research Cytogenetics Group (LRCG) and UKCCG Chair, Professor Christine Harrison, welcomed everyone to the 32nd meeting of the UKCCG. For the first time, the meeting was spread over two days and the UKCCG host, Leukaemia & Lymphoma Research (formerly the Leukaemia Research Fund) had kindly funded overnight accommodation as well as travel expenses for a delegate from each contributing laboratory. Christine also acknowledged the support and contribution of local groups, including the Centre for Haemato Oncology and the North East Children s Cancer Research Fund. Christine listed the recent publications that had included UKCCG members before giving an update on the ongoing UKCCG projects: Complex karyotypes in AML The karyotypes of 729 children and 5,876 adults with AML had generated a classification of AML into 54 cytogenetic categories. An analysis of survival has now been undertaken. There is no consistent definition of what constitutes a complex karyotype; some studies define it as having at least three abnormalities; others at least five. For this analysis, a complex karyotype was defined as having four or more abnormalities. The analysis showed that karyotype complexity had no impact on survival in children. High hyperdiploidy in AML is less frequent than it is in childhood ALL and it remains to be decided whether it should be classed as complex or not. Some patients with complex karyotypes have had acgh profiles prepared, and these are being analysed. Cytogenetics is being used in the new MRC Trial, AML17 as the basis of a risk score which includes age and other clinical features. The cytogenetic classification is based on the original publication from AML10 which is now ten years old, which reported on a very limited range of cytogenetic abnormalities. The risk score classification will be updated to include the newly described poor risk abnormalities. Intrachromosomal amplification of chromosome 21 (iamp21) Ongoing studies towards the further characterisation of iamp21 include genome wide array CGH, FISH, mutation screening, analysis of clustered breakpoints and deep sequencing. There is collaboration with an international study (Childhood ALL Working Group, Ponte de Legno ) to examine cytogenetics and clinical features. Other group This group (which is defined as excluding high hyperdiploidy, near haploidy, low hyperdiploidy, and all the established chromosomal abnormalities) is being studied with array CGH, SNP arrays and micro RNA expression. CRLF2 and IKZF1 There is clinical interest in these genes. They have been investigated in a FISH screen of 1000 cases of childhood B cell precursor ALL, and an MPLA kit (MRC Holland) to try to establish their incidence, their association with other chromosomal abnormalities, and their prognosis (see later presentation by Lisa Russell). The LRCG is in the process of screening >460 Ph negative adult ALL patients from UKALLXII and is preparing to screen 1500 children from ALL New studies: 1 ALL 2003 (B Cell Precursor (BCP) ALL): MLPA is being used to look for deletions in genes that are involved in B cell differentiation and cell cycle control. High throughput sequencing for mutations, and 60K customised arrays are planned in association with OGT (Oxford Gene Technology; see later). The genes of interest include those involved in constitutive activation of RAS signalling, disruption of P53/RB1 signalling, and JAK family members. These results will be used to inform the new ALL Trial, ALL 2010.

2 New studies: 2 In addition to T ALL, T NHL patients will be included in the new childhood ALL trial, ALL DNA from these patients will be studied by MLPA for known gains and losses, supplemented by FISH. These include deletions of TAL1, NF1, PHF6 (Xq), LMO2, CDKN2A, and 6q; gains of MYB and NUP214 ABL; and translocations involving TCR and TLX3. A Pilot study of DNA from 48 patients from ALL 2003 is being undertaken by Claire Schwab and Nick Bown. New studies: 3 In relation to childhood B non Hodgkin lymphomas: the previous FAB collaborative study has been published (Poirel et al, see above) and a new international trial is being set up. Christine will be contacting UKCCG labs about what data will be needed for this. The UK CCLG cell bank has 40 samples that will be examined by array CGH as a pilot study. Christine announced that the LRCG had been successful with an application to the European Research Council for funding to study Oncogenomics. This amounted to 2.25 million Euros over five years to develop strategies for high throughput in vivo screening in acute leukaemia. This work will be undertaken by Paul Sinclair with a technician and a PhD student. Finally, Christine thanked the UKCCG members for their continuing support and for providing the essential material that is needed for the LRCG research work. Dr Jan Cools. (Department of Molecular and Developmental Genetics, K.U.Leuven & VIB, Leuven, Belgium.) Molecular Genetics of T ALL: implications for therapy. The presentation began by summarising the genetics of T ALL, and the known functions of some of the genes involved. Chromosome translocations table: T cell receptor alpha 14q11 TLX1, TAL1, LMO1, LMO2 T cell receptor beta 7q34 q35 TLX1, LYL1, TAL2, NOTCH1, HOXA T cell receptor gamma 7p15 none T cell receptor delta 14q11 TLX1, TAL1, LMO1, LMO2 t(5;14)(q35;q32) / BCL11B translocations involving 11q23 t(10;11)(p13;q14) TLX3, NKX2 5 MLL CALM AF10 Point mutations: Some are activating, e.g. NOTCH1 (55%), JAK1 (10%) and FLT3 (5%) And some are loss of function, e.g. FBXW7 (20%), PTEN (20%), and WT1 (10%) JAK1 is subject to many mutations, all leading to activation; some associated with AML and some with ALL, unlike JAK2 (3 mutations) and JAK3 (3 mutations) The partner genes are associated with particular functions: (a) Cell cycle defect: p16 deletion / mutation (b) Differentiation block: TLX1, TLX3, LYL1, HOXA, MLL, CALM AF10, TAL1, TAL2, LMO1, LMO2 (c) Proliferation / survival: NUP214 ABL1, LCK, JAK1, FLT3 (d) Stem cell / T cell specification: NOTCH1, PTEN, FBXW7

3 Many cases have one from each of these groups, progressively (e.g. NOTCH1 > p16 > TLX1 > NUP214 ABL1) and a single oncogene is not thought to be sufficient to develop T ALL. In the normal state, NOTCH1 starts in plasma membrane; with the action of a NOTCH ligand and gamma secretase, it is cleaved and goes into the nucleus. The mutated form does not need the ligand to be cleaved, but needs only the gamma secretase. Therefore, this enzyme is a therapeutic target. However, using a gamma secretase inhibitor slows growth but does not halt it. Combined treatments are more effective, so adding Imatinib (a tyrosine kinase inhibitor), has a much greater effect: this acts on the proliferation / survival function of NUP214 ABL1. However, the precise actions of many genes remain to be determined. Modern techniques are adding to our knowledge of the genetics of T ALL. High density oligo arrays have found MYB to be duplicated in 8% of cases. MYB (at 6q23) is a nuclear transcription factor involved in the proliferation, survival and differentiation of haemopoietic progenitor cells. sirna can be used to knock down MYB, and this induces differentiation. PTPN2 (at 18p11) is a recurrent site of deletion in T ALL. Of 200 cases studied, five had mono allelic loss and eight had bi allelic loss. Twelve of these cases were TLX1 positive and four were NUP214 ABL1 positive. PTPN2 is involved in the JAK STAT signalling pathway as a negative regulator. Its loss results in a decreased sensitivity to Imatinib. T ALL has been found to have complex inter relationships between several genes, and the clinical implications of these are still being elucidated. There is the beginning of an understanding of the interreactions of these with potential targeted therapeutic agents. Lisa Russell (Leukaemia & Lymphoma Research Group, Newcastle) CRLF2 expression in BCP ALL The IGH@ gene normally produces the heavy chain of an antibody molecule. It is characteristically subject to somatic hypermutation, class switch recombination, and rearrangements of the V(D)J region, all of which involve double strand DNA breaks. It has been translocated with at least 45 partner genes, mainly in mature leukaemias and lymphomas. It has rarely been reported in B cell precursor ALL, and Lisa has been studying some of these rare cases. Her studies have included metaphase FISH, cloning of DNA breakpoints, RT PCR, and retroviral transfections (see publications mentioned earlier). One of the recurrent translocations is with the CRLF2 gene (cytokine receptor like factor 2) that is located in the pseudoautosomal region (PAR1) of the X & Y chromosomes. CRLF2, a type I cytokine receptor, heterodimerises with IL7RA and upon binding of the ligand, TSLP, activates the JAK STAT pathway. The activating tyrosine kinase remains unknown. There are 33 of these translocations in the database (9 being contributed from collaborators), 23 being known to be t(x;14)(p22;q32) and six being known to be t(y;14)(p11;q32). These translocations have been found in patients aged from 3 to 76 years (median 16). Six of these 33 had Down syndrome (18%). Array CGH (244k platform) was used to look at copy number changes, and this found frequent deletions of PAX5, IKZF1, and CDKN2A/B. Gain of X was also common. 64 cases were found that had a deletion of PAR1, centromeric of CRLF2, instead of a translocation. The age range was 1 to 35 years, median 4 years. 35 of these 64 had Down syndrome. Both the IGH@ translocation and the deletion within the PAR1 was associated with over expression of CRLF2, presumably due to the IGH@ enhancer and the promoter of the purigneric gene, P2RY8, respectivley. This deregulated expression induces proliferation of early B cell precursors by activating the JAK STAT pathway. These associations were assessed by testing on samples from ALL cases (B ALL and T ALL) were screened by FISH and 52 tested positive, all in BCP ALL. Nine had a translocation, and 43 a PAR1 deletion. Of these 52, 14 had Down syndrome, six had high hyperdiploidy, five had iamp21, seven had abnormality of 9p,

4 and 18 had an IKZF1 deletion. The survival of DS patients with CRLF2 deregulation was worse than the fewer cases without deregulation, but the numbers were too small to be reliable. There was no obvious effect on survival for non DS patients. It may become important to screen for CRLF2 as it may be a target for treatment. Although it is deregulated in only 5% of childhood ALL, it is deregulated in 52% of Down syndrome ALL. CRLF2 is the first gene in the PAR1 to be recurrently involved in leukaemia by translocation and interstitial deletion. Chris Bacon (Northern Institute for Cancer Research. Newcastle University & NHS FT.) Recent Advances in the genetics and biology of lymphoma. There are currently 11,000 new cases of lymphoma per year in the UK, and the incidence is increasing. The two commonest kinds (in adults) are diffuse large B cell lymphoma (DLBCL, 40%) and CLL/SLL. DLBCL occurs at all ages but the median is years. In 40% of cases, the disease is extranodal; in 50% the disease is advanced at diagnosis. Current treatment is R CHOP, which has a 40% cure rate. DLBCL is derived from germinal centre (GC) B cells. Germinal centres are areas within a lymph node where naïve B cells meet antigens and undergo somatic hypermutation. The progress of a lymphocyte towards becoming a mature plasma cell, and its functionality, are both controlled by a variety of genes, including BLC6 and PAX5, NFKappaB, IRF4, Blimp1 and XBP1. DLBCL is not a single disease: the WHO recognises a dozen categories plus DLBCL NOS (not otherwise specified). Gene expression profiling had indicated two main types, one in which the B cells are still GC like (GCBC), and another in which they are activated (ABC), appearing as plasmablasts, i.e. with terminal differentiation so that somatic hypermutation has ended. These two classes also have differing cytogenetic abnormalities: BCL2 is involved in GCBC (34%) but not in ABC; trisomy 3 is more common in ABC; translocations of BCL6 are also more common in ABC (24% vs 10%) but mutations are not (44% vs 74%). Abnormalities of TP53 are similar in both. The BCL6 mutations prevent IRF4 binding to cells blocked at the GC stage, and this results in defective differentiation. Acting at a different stage of the pathway, abnormality of Blimp1 (PRDM1) has a similar result. Almost all ABC DLCL express IgM. The ABC class of DLBCL has a worse prognosis with R CHOP, being about 50% at five years, compared to about 80% for GCBC. However, a new agent, Bortezomib, is effective, while being unsuitable for GCBC type DLBCL. Sporadic Burkitt lymphoma (BL) is generally well characterised (being MYC+ve and BCL2 ve), but many lymphomas appear to be in a grey zone between this and DLBCL, and are sometimes called BL like. The WHO describes the BL like class as BCLU IDB, meaning B cell lymphoma unclassifiable intermediate between DLBCL & BL. These diseases are morphologically very similar, but distinguishing between them is important for ensuring the correct treatment: ALL type for BL, and R CHOP for DLBCL. Molecular profiling classifies many cases, but still some remain unclassifiable. Paradoxically, some cases that fit the molecular profile of BL are BCL2 positive, and 10% are MYC negative. Cases with simple clones including MYC were most common in BL; complex clones including MYC were more common in the intermediatediagnosis group, and these have the worst prognosis. Complex clones occur in 71% of DLBCL but rarely involve MYC. (In this context, complexity was defined as having more than 6 abnormalities.) The MYC+ve DLBCL had an inferior prognosis. Classification by profiling has been shown to be beneficial in terms of providing the correct treatment. Some patients have a double hit, with abnormalities of both MYC and BCL2. This combination is associated with adverse clinical features, and the survival for this disease is very poor, with a median of less than 12 months. This presentation generated considerable discussion. In these lymphomas, clone complexity is a significant factor, and one that can be missed if only limited FISH studies are done. Profiling is not yet possible for most cases. A karyotype study of metaphase cells can provide this information, but often the material sent for cytogenetic study is inappropriate, being BM that is not infiltrated. However, if clinicians are made aware that it is important, then it is likely that more effort will be put into providing primary tissue.

5 Claire Schwab (Leukaemia & Lymphoma Research Group, Newcastle) MLPA assay in BCP ALL As gene copy number abnormalities (CNA) have been found to be common in BCP ALL, the LRCG wanted to identify a reliable technique for high throughput analysis of large numbers of samples for the presence of these abnormalities. Array CGH and SNP arrays are expensive and time consuming; FISH is also labour intensive and can lack sensitivity. However, MLPA can screen ~50 sequences in a single test at a higher resolution that is possible by FISH. The process involves the use of primers and stuffers, which are different for each probe. After denaturation and hybridisation, a ligase is used to join bound probes that are extremely close to each other. PCR is used to amplify the ligated probes, and the amount of product is proportional to the copy number. The products are separated by capillary electrophoresis and the results are compared with controls. MRC Holland has manufactures a kit with the probes that the LRCG had identified as being potentially significant. These included probes for IKZF1 (IKAROS), CDKN2A/B (p16), PAX5, ETV6, RB1, BTG1 (12p), EBF1 (5q) and PAR1 (CRLF2). The technique was tried first on material (fresh and/or fixed) from 43 high risk patients who had already had a variety of FISH studies. In most cases there was concordance between FISH and MPLA results, with MPLA being more successful in detecting CDKN2A loss. In a few cases where there was low level of infiltration, the FISH was more successful. The kit is now being used on adults in UKALLXII, with 250 already studied and over 1000 to go. A kit for T ALL is being designed. Nick Cross (Wessex Regional Genetics Laboratory, Salisbury) New genetic findings in myeloproliferative neoplasms (MPN) MPN are chronic conditions, including chronic myelogenous leukaemia (CML), polycythaemia vera (PRV), primary myelofibrosis (MF), essential thrombocythaemia (ET) as well as rarer conditions such as atypical CML, chronic myelomonocytic leukaemia (CMML) and chronic eosinophilic leukaemia (CEL). Excluding BCR ABL1 fusion positive CML, only about 25% of MPN have cytogenetic abnormalities, and most of these have a simple trisomy 8 or 21. Monosomy 7 is seen in a few cases, as are deletions of 7q, 13q and 20q. Translocations, mostly producing tyrosine kinase fusions, are found in 2%. Nick has details of 48 translocations, and these involve a few recurrent genes (such as PDGFRB at 5q33 and FGFR1 at 8p11) with a wide variety of partner genes. It is important to identify these cases, as they can be treated with Imatinib; all are cytogenetically visible except one, FIP1L1 PGFRA. Equally, there are now targeted therapies available for those patients who have other genes involved, such as JAK2 or FLT3. Acquired uniparental disomy (AUPD, also called acquired isodisomy) is being detected in many haematologic disorders, so SNP arrays have been used to investigate 148 MPN cases that had apparently normal chromosomes. 63 tracts of greater than 20MB were found, mostly in 4q, 7q and 11q. aupd at 4q and 11q is associated with TET2 and CBL mutations, respectively. There are several hundred genes within the minimal 7q region but dense acgh and Agilent tiling array has found a 3 gene target that is the subject of a submitted publication.

6 Genetic predisposition to MPN. There are rare families with multiple cases of MPN, and it as been calculated that the sibs of someone with MPN have a five times risk of developing a similar disorder. There is no evidence of JAK2 V617F being inherited, yet it occurs in 95% of PRV and 50% of ET. A particular constitutional haplotype, 46/1, which was found to occur in 24% of 1,500 UK blood donors, predisposes to MPN (O/R for carriers = 3.7). There are two main hypotheses about the association between 46/1 and MPN: (a) V617F may preferentially arise on a 46/1 haplotype due to a specific mutational mechanism; (b) the probability of clinical MPN may be higher if V617F arises on 46/1. Nick said that he marginally favours hypothesis (b), which is supported by the observation that similar results are found with Crohn s disease. However analysis of patients entered into the UK PT 1 trial found no association between 46/1 and haematological features or clinical outcome. 46/1 is the first known instance of a constitutional risk factor that is associated with specific somatic mutations, and it remains to be determined precisely why it predisposes to MPN, and whether other acquired mutations are also associated with specific haplotypes.

7 Tuesday 16 th March. Anthony Moorman (Leukaemia & Lymphoma Research, Newcastle): Trials & Data Analysis Update. In the last 17 years, the group has collected data and/or samples from 26,659 patients, which form the basis of its research work, practical and statistical. Current Trials are: New Trials are: The ALL2003 Trial ends in 2010; it has 2607 entries, and there are 89 with data still awaited. The EsPhALL Trial has 43 patients; it is scheduled to end in The UKALLR3 Trial will end 2011 and will be replaced with an International ALL relapse, for which the UKCCG will be collection centre. The Interfant Trial will continue until The AML16 Trial has accrued 1709 entries; its intensive arm is closed but it is still open for the nonintensive arm. ALL2010: due to start in December Regimen C will include patients with high risk cytogenetic abnormalities, including near haploidy, low hypodiploidy (30 39 chromosomes), iamp21, t(17;19)(q23;p13), and MLL translocations, for which the partner must be identified. Ph+ve cases will be transferred to EsPhALL. UKALL14: due to start in Summer It will include an older age range than UKKALLXII (25 65 instead of 15 60). Anthony will circulate full details of the cytogenetic requirements for this trial when it opens. There are four known high risk groups: Ph+ve, t(4;11)(q21;q23), Low hypodiploidy (30 39 chromosomes) / near triploidy (60 78 chromosomes); and complex karyotype. Patients with these abnormalities should go for stem cell transplant, using unrelated donors if necessary. However, Ph+ve cases will be treated immediately with Imatinib, and so its presence needs to be identified urgently. Fails and normals need to be screened with MLL, and the Newcastle group will, if necessary, look for AF4 involvement in MLL+ve cases. Complexity is defined as 5+ abnormalities in the absence of an established ploidy group. Normal / fail cases will need to be screened either by DNA cytometry or by FISH to look for low hypodiploid and near triploid clones. It is recommended that probes are used for chromosomes 1, 6, 11, & 18 and 3, 7, & 15. (See Charrin et al (2004) Blood, 104, 2444). Outcome of closed Trials: ALL97 & ALL99 were two phases with different risk stratification criteria, but the high risk cytogenetic classification was consistent. The overall outcome was 81% RFS at 5 years, with the best groups being ETV6 RUNX1 (89%) and high hyperdiploidy (86%), and the worst being iamp21 (27%), near haploidy (44%), low hypodiploidy (50%), MLL involvement (55%) and Ph+ve (56%). Only two patients had the notorious t(17;19)(q23;p13) and both have relapsed and died. Most of these remained highly significant in multivariate analysis; MLL did not and this may be because of an association of t(4;11) with older children. The composition of the high hyperdiploid group was assessed: there was no difference between those cases with gains of 4, 10 and 17 compared to those without; however, cases with gain of 18 fared substantially better. Abnormalities of 17p had little adverse effect when they occurred in ETV6 RUNX1 or high hyperdiploid clones, but were otherwise poor. Loss of 13q did not have a major effect on overall survival, though it was associated with a shorter EFS. The prognostic information from cytogenetic studies extends into outcomes after relapse, with 92% of good risk cases alive at 7 years, 64% of intermediate risk cases, and only 14% of poor risk cases being alive at a maximum of six years (so far). These results have now been published: Prognostic effect of chromosomal abnormalities in childhood B cell precursor acute lymphoblastic leukaemia: results from the UK Medical Research Council ALL97/99 randomised trial. Moorman AV, Ensor HM, Richards SM, Chilton L, Schwab C, Kinsey SE, Vora A, Mitchell CD, Harrison CJ. Lancet Oncol May;11(5):429 38

8 Phil Stephens (Cancer Genome Project, Sanger Institute) Characterisation of structural variation in breast cancer genomes using paired end sequencing on the Illumina genome analyser. Genetic studies of cancer are identifying new targets for anticancer treatment, and will be essential in an era of personalised treatment. Unlike the situation in leukaemias, where a clone with five abnormalities would be described as complex, solid tumours usually have many times more than this. About 20 cancer causing mutations are known, and it is thought that there might be 100,000 passenger mutations. With this level of complexity, there has been pessimism about finding a simple, effective therapy. However, this pessimism may be unfounded. For example, the BRAF V600E oncogene is involved in 70% of melanomas. This was dismissed as being an unsuitable target, yet two years ago a new drug, Plexxicon 4032, which is a selective inhibitor of BRAF, was found to have a dramatic effect in only 15 days, measured radiologically. It may have cleared 99% of the tumour cells, which leaves only 1% needing a new treatment target to be identified. The Illumina analyser works on sheared DNA that has been ligated to Illumina adaptors, then amplified and sequenced. The sequencing technology has progressed so much that what would have taken years to do now takes a single day. Therefore it is possible to screen for commonly occurring somatic mutations, and compare acquired abnormalities with the germline. The breast cancer cell line HCC38 was found to have 238 somatic structural variants, the great majority being tandem duplications. Other cell lines were found to have different pattern of abnormality types. Fifteen primary breast cancers have also been studied; 1014 somatic structural variants were found, ranging from 231 in one case, while another had only seven and one, an invasive lobular carcinoma, had only one. Findings so far include ten in frame fusion genes; previously only one was known. 139 genes were rearranged in more than one sample. 22 gene fusions have been exome sequenced using Agilent Sure Select. Two novel pathways have been found to be deregulated in a large proportion of ER positive breast cancers. This technique has potential applications in health care. Solid tumours shed cells into the bloodstream, and quantitatively detecting these cells with tumour specific mutations could be used for monitoring response to different treatments, detecting relapse, etc. This can save on the cost and harmful effects of ineffective treatments. Three wealthy patients have already used the technique and had their treatment modified by the results. Identifying the tumour specific rearrangements takes about two weeks and costs about 2.5k, and extracting the DNA from blood and quantifying it takes a day and costs about 100. A grant of 1.7m has been obtained to study 100 cases. Olaf Heidenreich (Northern Institute for Cancer Research) Fusion genes as potential therapeutic targets Survival rates for paediatric leukaemia have improved from 10% in the 1950s to nearly 90% today. Much of this has been due to better use of drugs. However, successes in other cancers have been too rare. Leukaemias lead the way because of having simpler and more stable genetics and easier access to tissue for study. Current therapies don t distinguish between normal and cancer cells, and the ideal would be to target the cancer cells. This is becoming possible with new treatments that are specific for acquired genetic abnormalities. sirnas (small interference RNA) were designed to fit fusion sites for translocations such as t(8;21) and t(4;11). Of these, the siaml1/mtg8 impaired crystal cell formation in drosophila, but was less effective when tested

9 on mice. Surrogate targets are sought, and these have included telomerase, Angiopoietin ANGPT1, and Calpains. In order to directly target fusion genes a therapeutic sirna approach is being developed sirna rapidly degrades (1 2 minutes). Therefore it has been encapsulated, and a ligand has been added to target the leukaemic stem cell. It has been found that uptake is associated with an effect on transcription and it decreases clonogenicity. Jon Strefford (University of Southampton, School of Medicine) Genome wide genetic analysis of chromosome abnormalities in CLL CLL is a disease with an extremely heterogeneous course: some patients have an aggressive disease and die within six months; others have an indolent disease that can last for more than twenty years. CLL has traditionally been classified at diagnosis by a staging system; however, this gives very little information about the likely prognosis. Cytogenetic studies have indicated outcome: Loss of 17p (TP53) 5 7% of CLLs have loss or mutation of TP53. In patients needing treatment, this has been associated with a poor prognosis, and with no response to standard therapy. Loss of 11q (ATM); 10 20% of CLLs; 30% have mutations in the remaining ATM gene. Prognosis is poor, but this doesn t currently affect type of therapy. Loss of 13q (40% single; 10% homozygous). As the sole abnormality, this has been regarded as being an indicator of a good prognosis. Jon has been using an Affymetrix SNP6.0 array platform on CLL material from a variety of sources, including the CLL IV Trial. This technique found acquired genomic imbalance (mostly deletions) in 94% of cases, with up to 25 somatic alterations. It was found that the 13q deletion varied widely in size, with a consistent 2MB region, and that the cases with progressive disease were more likely to have larger deletions. The association between size and prognosis was independent of factors such as IgVH mutation status, ZAP70 expression, and other cytogenetic abnormalities. Sequential studies of one case found progressive increase in the size of the deletion. It was found that most 11q deletions were large, so it is unlikely that only the ATM gene is involved. Some cases had acquired uniparental disomy (aupd). Similarly, deletions of 17p were mainly large. Some other recurrent sites were found, with gain of 2p, and losses of 13p and 18p. Fiona Ross (Wessex Regional Genetics Unit, Salisbury) Myeloma IX genetics: Results of a 3 year interim analysis Fiona gave an update on the genetics of myeloma and the first official analysis from the MRC Myeloma IX trial. Many cases are hyperdiploid, with gains of mainly the odd numbered chromosomes. Some of these also have translocations of IGH@, but IGH@ is most commonly involved in non hyperdiploid clones. The international myeloma working group has advised that that all patients should be tested for the abnormalities that are known to be associated with a poor prognosis on conventional therapy. These are t(4;14)(p16;q32), t(14;16)(q32;q22) and del(17)(p13). Also associated with a poor prognosis are t(14;20), 1q gain / 1p deletion, and possibly 12p deletion. 13q deletion is no longer thought to have independent poor prognostic value, and previous findings are now known to be due to its strong association with t(4;14),

10 t(14;16) and t(14;20). Hyperdiploidy and t(11;14)(q13;q32) are associated with an intermediate prognosis, and gain of 5q may indicate a better prognosis (p=0.037). To be sure of detecting the clinically significant abnormalities, it is essential to do FISH on either identified plasma cells (usually by simultaneous immunostaining) or on samples that have been plasma cellconcentrated by CD138 cell selection. This is because a high proportion of samples will be seriously haemodilute and contain a very much lower proportion of plasma cells than seen on the morphology slides. Just over 1000 Trial patients have been studied by Fiona, primarily with FISH but also by karyotype where there was enough material. The FISH was batched, with nine micro dot tests per slide. The results included the following: hyperdiploidy 58%; 13q 45%; IGH@ rearrangements 44%; 1q gain 39%; 1p 11%; 22q 13%; and 17p 8%. Only 8 cases (<1%) had t(6;14), and 16 (1.5%) had t(14;20). t(4;14) was 11% and t(11;14) 14%. Several of the abnormalities tended to co occur. The association between 13q and the poor risk IGH@ translocations has been mentioned, but it also occurs with 17p and gain of 1q. Patients with bad IGH@ translocations fared significantly worse than the rest in both the intensive (p=2.04x10 6 ) and the non intensive (p=5.35x10 7 ) treatment arms of the trial. The co occurrence of certain cytogenetic abnormalities makes it difficult to determine their individual effects, but bad IGH@ and 1q gain are independent and had a worse impact on outcome when present together than when only one is present. The MM IX Trial confirmed the PFS benefit of maintenance thalidomide at all ages, but particularly for older patients who cannot take aggressive treatment and SCT. However, PFS was not improved for patients in the adverse cytogenetics group and OS was significantly worse. The next myeloma Trial, Myeloma XI, is being planned. Regretfully, karyotype studies will not be part of it, although extensive genetic results will be collected. As there is a randomisation to Velcade in this trial it should help to clarify whether this drug can overcome the poor prognostic effect of t(4;14) patients. Nick Bown (Newcastle) introduced some contributions from UKCCG members. Mustafa Daghistani (Department of Haematology, Imperial College at Hammersmith Hospital, London) EVI 1 oncogene expression predicts survival in chronic phase CML patients resistant to Imatinib. The Philadelphia translocation, pathognomic for chronic myelogenous leukaemia, produces a fusion of the BCR and ABL1 genes, which results in deregulated tyrosine kinase activity. This has become the target for new therapies and the use of tyrosine kinase inhibitors (TKIs), such as imatinib, has had a dramatic effect on survival. However, in long term use, 30 40% of patients show resistance or intolerance to imatinib. Second generation TKI agents, including dasatinib and nilotinib, are effective in up to 60% of imatinib resistant cases, however resistance remains a clinical problem. The mechanisms of resistance are being studied; these vary from the presence of a particular mutation, T315i, which renders dasatinib and nilotinib ineffective, to the activation of pathways that are alternative to BCR ABL1, although the role of the latter is poorly understood. The search is therefore on for clear evidence of signal transduction pathways whose activation may render CML cells BCR ABL independent and thus refractory to TKI. One being investigated at the Hammersmith is EVI 1, well known to cytogeneticists as being involved in the abnormalities of chromosome 3 that are associated with dysmegakaryopoiesis in AML. These include inv(3)(q21q26) and t(3;21)(q26;q22), however EVI1 activation in the absence of overt 3q26 rearrangement has also been demonstrated in a range of myeloid neoplasms. The group at Hammersmith had previously found 3q26 abnormalities in patients who are resistant to the second generation TKIs, and therefore looked for EVI 1 expression in a prospective series of 75 imatinib resistant chromic phase CML patients. None of these had visible abnormality of 3q26. Expression was measured with a multiplex TaqMan assay, and 8/75 cases proved positive. A retrospective study of the diagnostic samples of the same eight found no expression at that time, indicating that expression had been acquired during the course of the disease.

11 Importantly, these eight cases had a significantly poorer PFS, EFS and overall survival than other patients with imatinib resistance. In a multivariate analysis, EVI 1 expression status and the best cytogenetic response obtained on imatinib were the only independent predictors for OS, PFS and EFS. These data suggest that screening for EVI 1 expression at the time of imatinib failure may predict for response to second line TKI therapy and consequently aid clinical management; such cases might be better treated with an early stem cell transplant. Isabel Boyd (Duncan Guthrie Institute of Medical Genetics, Royal Hospital for Sick Children, Glasgow) An unusual case of Infant ALL with a complex MLL rearrangement and a concomitant Burkitt rearrangement. An 11 week old infant presented with pallor, lethargy, and 93% blasts that had an immunophenotype positive for CD10, CD19 and HLADR. The karyotype was 46,XX,t(7;19;11)(q11.2;p13.3;q23) [2] / 46,idem,t(8;22)(q24;q11) [6] / 46,XX[2] FISH studies confirmed involvement of both MLL at 11q23 and MYC at 8q24. The child was deemed to have common ALL with CNS involvement, was treated on Interfant 06, medium risk group, and the response so far has been good. Mike Evans (Oxford Gene Technology) From large scale research to cytogenetic monitoring an integrated platform for CNV analysis. 5% of the human genome is subject to CNV (copy number variation) and in the last five years there has been a big increase in publications, mainly on constitutional genetics. These have linked CNV to conditions such as obesity and schizophrenia. However, most diseases are complex, and linking them to genes requires detailed studies of many samples. OGT has completed CNV analysis of 20,000 samples from the Wellcome Trust Case Control Consortium. This required advanced technology not only for the sample preparation and the genetic analysis but also for needs such as the tracking of samples and the data analysis. Much of the past work has been in constitutional genetics, and OGT is keen to expand into cancer. It would welcome samples sent for study, and will design probes according to customer requirements. Mervyn Humphreys (Northern Ireland Regional Genetics Centre; Belfast City Hospital) Case Report: A patient with AML and MDS related changes, showing a complex karyotype including extensive telomere association A 74 year old man was referred with a mild asymptomatic anaemia. A cytogenetic study of bone marrow found five divisions from a complex clone: 45,XY, 1,der(5)t(5;12)(q13;q13),add(9)(p?), der(11)t(11;17)(p11.2;q2?1), 12, der(17)t(1;17)(p1?3;q21), +der(17)t(1;17)(p1?3;q21), add(18)(p11.2) At this time, the BM morphology showed trilineage dysplasia with no excess of blasts. He had progressive fall in platelet counts during the next nine months. At this time he had developed fever, epistaxis and severe lethargy, and his BM morphology showed transformation to AML. A cytogenetic study found a near triploid clone with some of the abnormalities that had been seen previously. However, on this occasion most of the

12 clonal metaphases had pairs of chromosomes with telomere association. Twenty different pairings were seen, one of which, between 15p and 22p, occurred in three cells. Telomere association is relatively rare but has been reported in several different solid tumour types and LPD; it has not been previously reported in a myeloid malignancy. Its biologic and clinical significance is unknown. Mervyn asked the audience if anyone had a similar case. A few cases have been seen with telomere association, but not at this high level. Emma Huxley (West Midlands Regional Genetics Laboratories, Birmingham) Case report: A case of CML with granulocytic sarcoma A 40 year old man had presented with a granulocytic sarcoma and a WCC of 220. Cytogenetic analysis of blood sample confirmed a diagnosis of CML, with a karyotype 46,XY,t(9;22)(q34;q11)[10]. RT PCR was positive for a b3a2 BCR ABL1 gene fusion. He was started on Imatinib. A cytogenetic study of blood done ten weeks later found one normal division and one with a karyotype of 48,XY,t(8;21)(q2;q2), t(9;22)(q34;q11),t(18;19)(q2;p13),+der(21)t(8;21),+der(22)t(9;22). A FISH study confirmed a RUNX1 RUNX1T1 dual fusion. A retrospective study of 60 metaphases from the diagnostic sample found two with the t(18;19), and five with +8, but none with t(8;21). However, low levels of RUNX1 RUNX1T1 fusion were found by FISH and RT PCR. A BM study at 3 months showed no evidence of response to Imatinib. More divisions were seen with the t(8;21) and +der(21). Treatment was changed to Dasatinib; there appeared to be an initial response, but this was not maintained. The cells with t(8;21) became predominant. AKD mutation analysis was instigated and this found an F317L mutation. This is known to be associated with resistance to Dasatinib, but with sensitivity to Nilotinib and partial sensitivity to Imatinib. At this stage, the man was considered to have developed AML, so chemotherapy was started. After three months, a cytogenetic study of 60 divisions found ten divisions with just a t(9;22) and only one division with the additional t(8;21). The granuloma in this man was deemed to be an extramedullary blast phase. There have been only three previous publications of cases with concurrent t(9;22) and t(8;21). Emma highlighted that the karyotype and FISH studies had frequently shown large differences in the proportions of clonal interphase and metaphase cells. Paula Waits (Bristol Genetics Laboratory). The development clonality testing for lymphomas in the Bristol Genetics Laboratory (BGL). In her introduction, Paula stated that up to 95% of lymphoid malignancies are of B cell lineage, with up to 7% being of T cell lineage. >98% of these contain clonal rearrangements of the immunoglobulin gene or T cell receptor genes. These genes have an enormous capacity for variation and diversity, known as somatic hypermutation, which is necessary for their normal function in identifying antigens. The rearrangements include deletions, insertions, and mutations, and they occur in mature cells. Divisions of these cells result in clones that are not malignant. Many of these clones are too small to detect.

13 In 2009 the BGL introduced clonality testing using the Identiclone kits from InVivoScribe Technologies using an array of different samples types including peripheral blood, solid tumour and paraffin embedded tissue. Paula described the results from four cases in which the presence of a malignant clone was identified using an algorithm: the peak has to be at least three times the height of the third highest peak in the normal, polyclonal background. Norman Pratt (Human Genetics Unit, University of Dundee) The wages of CIN (chromosome instability): A CGH in colo rectal cancer Colorectal Cancer is the third most common in the UK, with 37k new cases per year and 16k deaths. Like most cancers, it is a disease of old age. It arises from premalignant polyps in the bowel, and there is a national screening project for early detection. About 25% of cases have a familial link, and in some of these BRCA1, BRCA2 are implicated. Some genes are known to be associated with progression of the disease: mutation of APC (at 5q21) is an early first hit event; K ras (at 12p12) is implicated between adenoma and large adenoma; mutation of TP53 and LOH of 18q occur with the development of carcinoma. By this stage the karyotypes are very complex, often being near tetraploid or higher. The results of array CGH studies of 50 tumours were shown. 80% had gain of 20q; 76% had loss of 18q; both of these were associated with high grade dysplasia and with mutations of TP53. Other recurrent abnormalities included 74% gain of 13q. Norman then showed the results of micro array studies, which have produced large quantities of detailed data. However, it would be clinically more useful to be able to identify confidently the disease at the adenoma stage, as a surgical cure is much cheaper than later chemotherapy, and is more beneficial for the patient. Therefore it is planned to micro dissect likely material from polyps and study this for potential markers for clinical use. Also, early genetic events are likely to be disease drivers, and therefore possible targets for new therapies. Steve Clifford (Northern Institute for Cancer Research, Newcastle) Childhood medulloblastoma: from genomics to improved therapies. Medulloblastoma (MB) is the cause of 10% of childhood cancer deaths; it is the most common type of brain tumour, and has a peak incidence at 7 years of age. Improving treatments has achieved a cure rate of 65%, but this is at a plateau. Also, surgery does serious damage to the brain. There is the need to optimise current treatments, minimise side effects, and discover new therapies. There was little biological information about MB up to 1998, and getting metaphases from fresh tissue for karyotype studies was particularly difficult. Some genetic information was obtained from family studies, such as those with Li Fraumeni syndrome (TP53 gene), Turcot s syndrome (APC), and Gorlin syndrome (PTCH1). Information about development signalling pathways is now being obtained, and these could be a drug target. The Sonic Hedgehog (SHH) pathway identifies a MB subgroup, as does Wnt/Wg. Expression arrays have found five genetic profiles More information was presented about the molecular characterisation of MB with activation of the Wnt/Wg pathway. 19 samples were studied by array CGH; all had characteristic monosomy for chromosome 6, but there was no evidence of the chromosomal abnormalities that are recurrent in other types of MB, such as those involving chromosomes 7, 9, 16 and 17.

14 The SHH pathway in combination with activation of COL1A2 is often associated with infant (<3y) desmoplastic MB, and these patients tend to fare well. The converse, SHH ve, COL1A2 ve, non desmoplastic MB has a poor prognosis. These genes were studied in the PNET3 Trial. (1992 to 2000, jointly between (SIOP) International Society of Paediatric Oncology & (UKCCSG) UK Children s Cancer Study Group). There were 109 informative cases; 25% had expression of beta catenin; 60%of these had a CTNNB1 mutation. These patients had a 92% ten year survival rate. 75% were negative for beta catenin, and did not have mutations of CTNNB1 and APC; these had a 65% overall survival at ten years. 178 cases from the PNET3 Trial and 114 other cases of MB were studied for MYC family amplification, which was indicated by a copy number of >5. MYC was amplified in only 4% of 263 cases and MYCN in 1%. Both were associated with a worse prognosis. Other high risk features included LCA and M2 3. These data can be used for stratification, so that good risk cases are not over treated. The favourable group (15%) are those cases with expression of beta catenin, no amplification of MYC, m0, and classic or desmoplastic histology. The intermediate group is the same except for no expression of beta catenin. The high risk group (30%) has amplification of MYC or MYCN, M+, large cell or anaplastic histology. In this group the beta catenin status is irrelevant. There is a feasibility study for 6 to 12 months, and if this is successful then it will be used in PNET5 and PNET6. Newcastle is the Trial co ordinating centre. Better diagnosis and more precise stratification are helpful, but new therapies are still needed. The drug companies have advised against targeting beta catenin. In mice the SHH pathway has been successfully suppressed by a small molecule inhibitor, HhAntag691, although there was a side effect on growth plate development. However, we are reaching point where genomics is starting to verge on therapeutic effect. Christine thanked all the speakers for their contributions to another successful UKCCG meeting. This year the meeting had been spread over two days, allowing time for more contributions a worthwhile format that may be used again next year. Meeting notes drafted by John Swansbury