Cytogenetics Update. Lynda J Campbell

Transcription

1 Cytogenetics Update Lynda J Campbell lynda.campbell@svhm.org.au

2 Nowell and Hungerford, 1960 Ph

3 Janet Rowley showed the Ph chromosome to be a balanced rearrangement: t(9;22) 9 22

4

5 Acute lymphoblastic leukaemia

6 Moorman et al, Blood, 2007 EFS of patients treated on UK MRC ALL97 by cytogenetic subgroup

7 4 year old boy presented with sudden onset of tiredness and bruising FBE showed blasts in PB t (12;21)(p13;q22) 46,XY,del(12)(p13)[22]

8 HEH karyogram

9 High hyperdiploidy in paediatric ALL Hyperdiploidy in acute lymphoblastic leukaemia associated with consistent pattern of trisomies and often four copies of chromosome 21 FISH for trisomies of 4, 10 and 17 ETV6/RUNX1 CEP4, 10, 17

10 t(4;11) seen in approx. 3-5% childhood and adult ALL - immature phenotype and often myeloid antigen High white cell count poor prognosis in both children and adults t(4;11)(q21;q23)

11 15-30% adult ALL but only about 3% childhood ALL either p190 or p210 fusion product seen associated with a dismal prognosis in both adults and children transplant candidates Some promising results with imatinib therapy Ph+ ALL

12 t(17;19)(q22;p13) with TCF3-HLF fusion t(17;19)(q22;p13) gives rise to the TCF3-HLF fusion It is a variant of t(1;19)(q23;p13), TCF3-PBX1 fusion. Very rare Extremely poor outcome in paediatric series Usually visible by cytogenetic analysis but may be confirmed using the dual colour breakapart probe specific for TCF3 (E2A) Report of t(17;19) in 23 adults showed no difference in overall survival compared with negative patients (Burmeister et al, Haematologica, 2010)

13 Multiple copies of RUNX1 clustered on a marker chromosome

14 intrachromosomal amplification of chromosome 21 (iamp21) 2% childhood ALL - pre-b immunophenotype, significantly older (median 9 years vs 5 years), lower white cell count (median 3.9 vs 12.4) significantly inferior EFS at 5 years : 29% versus 78% and overall survival 71% versus 87% respectively. 3-fold increase in relapse risk, New patients with iamp21 on UK MRC ALL2003 trial high-risk arm and considered for bone marrow transplantation in first CR.

15 Adult ALL Philadelphia status generally considered the most important cytogenetic indicator of outcome MRC/ECOG collaborative study of >1500 adults with ALL identified: Inferior outcome Ph translocation t(4;11)(q21;q23) Burkitt translocation* Complex karyotype ( 5) * Low hypodiploidy/triploidy * Moorman et al, Blood 2007 Improved outcome High hyperdiploidy Deletion of 9p * Independent of sex, age, WCC and T-cell

16 WHO 2008 classification of AML: AML with recurrent genetic abnormalities AML with t(8;21); RUNX1-RUNX1T1 AML with inv(16) or t(16;16); CBFB-MYH11 APL with t(15;17); PML-RARA* AML with t(9;11); MLLT3-MLL# AML with t(6;9); DEK-NUP214 AML with inv(3); RPN1-EVI1 AML (megakaryoblastic) with t(1;22); RBM15-MKL1

17 Cytogenetically Normal AML (CN-AML) Acute Myeloid Leukaemia with t(8;21); RUNX1- RUNX1T1 Figure 1. Pie chart illustrating the molecular heterogeneity of cytogenetically normal AML based on mutations in the NPM1, CEBPA, MLL, FLT3 (ITD and TKD mutations at codons D835 and I836), NRAS, and WT1 genes. Data are derived from mutational analysis of 485 younger adult patients with cytogenetically normal AML from AMLSG. Dohner et al, Blood, 115: , 2010

18 Acute Promyelocytic Leukaemia t(15;17)(q22;q21) PML/RARA fusion

19 Rare variants: t(11;17)(q23;q21) RARA/PLZF t(5;17)(q35;q21) RARA/NPM t(11;17)(q13;q21) RARA/NuMA interstitial del(17q) RARA/STAT5b PRKAR1A-RARA fusion in variant APL, Catalano et al, Blood 2007 PML/RARA probe RARA with PRKAR1A BAC probe

20 Inversion 16 AML M4Eo Fusion of CBFB on 16q22 and MYH11 gene on 16p13 AML with maturation with Auer rods & eosinophilia. Fuses the RUNX1 gene on 21 with the RUNX1T1 (CBFA2T1, ETO) gene on 8q22

21 Probe for inversion 16: CBFB/MYH11 dual fusion translocation probe (Cytocell Aquarius Probe)

22

23 MLL break apart probe (Vysis) to confirm t(9;11)

24 11q23 abnormalities Rearrangements of MLL observed in high-risk paediatric, adult and therapy-related acute leukaemias At least 104 different MLL rearrangements reported with 64 of the translocation partner genes

25 Commonest MLL rearrangements MLL partner genes MLL translocation Acute leukaemia AFF1/AF4 t(4;11)(q12;q23) 319/321 ALL MLLT3/AF9 t(9;11)(p22;q23) 41/125 ALL (mostly paed. 84/125 AML MLLT1/ENL t(11;19)(q23;p13.3) 72/87 ALL MLLT10/AF10 ins(10;11)(p12;q23) 40/54 AML MLLT4/AF6 t(6;11)(q27;q23) 28/35 AML ELL t(11;19)(q23;p13.1) 30/31 AML EPS15/AF1P t(1;11)(p32;q23) 7/13 ALL MLLT6/AF17 t(11;17)(q23;q21) 8/8 AML MLLT11 t(1;11)(q21;q23) 7/8 AML (all paediatric) SEPT6 ins(x;11)(q24;q23) 7/7 AML Meyer et al, Leukemia 2009

26 Overall survival curve for patients with 11q23/MLLrearranged pediatric AML grouped on the basis of different translocation partners t(1;11) t(10;11) Balgobind et al, Blood 2009, Vol. 114, No. 12, pp

27 Recent case: 23 year man presented with AML p13 (AF10) q14 (CALM) q23 (MLL)

28 FISH required to diagnose t(10;11) Type of rearrangement and breakpoints are variable. Observed in both ALL and AML A significant proportion result in an MLL-AF10 fusion (strongly associated with M5/M5a). Two other possible transcripts also described. CALM-AF10 (seen in both T-ALL and immature AML) Rarely MLL-ABI-1 (two cases reported)

29 Van Limbergen et al (2002) proposed 4 recombination patterns for the MLL/AF10: Type 1: inversion of MLL then t with 10p13. Type 2: inversion of MLL then ins into 10p13. Type 3: inversion of AF10 then t with 11q23. Type 4: inversion of AF10 then ins into 11q23. Morphologically, our case appeared to be a type 2 inversion / insertion but

30 MLL break apart probe: normal result assumed to be CALM-AF10 fusion formed by translocation

31 Prognosis categories in AML MRC % SWOG/ECOG % Good Inv(16)/t(16;16) ; t(15;17), t(8;21) +/- other abn; Intermediate Poor Normal, 11q23 abn, +8, del(9q), del(7q), +21, +22, all others Del(5q)/-5, -7, abn 3q, and Complex ( 5 unrelated abn) t(6;9)*, t(9;22)* 21 % 62 % 17 % Inv(16)/t(16;16) ; t(15;17)+/- other abn; t(8;21) without del(9q) or Cx Normal, +8, +6, -Y, del(12p) Del(5q)/-5, -7/del(7q), abn 3q, 9q, 11q, 20q, 21q, 17p, t(6;9), t(9;22) and Complex ( 3 unrelated abn) Unknown N/A All other abn 4% Ref: Slovak et al, Blood, 96: 4075, 2000; Grimwade et al, Blood, 92: 2322, % 46% 30%

32 Standardized reporting for correlation of cytogenetic and molecular genetic data in AML with clinical data Favourable Intermediate-I Intermediate-II Adverse t(8;21)(q22;q22); RUNX1-RUNX1T1 Inv(16) or t(16;16) CBFB-MYH11 Normal karyotype: Mutated NPM1 (no FLT3) Mutated CEBPA Normal Karyotype: Mutated NPM1+ FLT3 ITD Wild type NPM1+ FLT3 ITD Wildtype NPM1 + no FLT3 ITD t(9;11)(p22;q23); MLLt3-MLL Cytogenetic abnormalities not classified as favourable or adverse Inv(3q) or t(3;3); RPN1-EVI1 t(6;9)(p23;q34); DEK-NUP214 t(v;11)(v;q23); MLL -5 or del(5q) -7 Abnormality of 17p Complex karyotype: 3 abnormalities Döhner et al: Recommendations on behalf of the European LeukemiaNet, Blood 2010

33 Acquired uniparental disomy in CN-AML Bollinger et al, Leukemia (2010) 24,

34 FISH protocol for AML: FISH for t(15;17), inversion 16, t(8;21) or an MLL translocation if morphological or cytogenetic evidence to suggest the presence of a either a standard or variant aberration FISH for inversion 16 in all follow-up cases

35 IPSS for MDS: Survival and AML evolution Prognostic variable Score Value BM blasts (%) < Karyotype# Good Intermediate Poor Cytopenias* 0/1 2/3 # Good: normal, -Y, del(5q), del(20q) Poor: complex ( 3) or chromosome 7 abn. Intermediate: other abnormalities * Cytopenias: Hb <100g/L, Neutrophils <1.8x109/L, Platelets <100x109/L Ref: Greenberg et al, Blood 89: , 1997

36 WPSS for MDS Variable WHO category RA, RARS, 5q- RCMD, RCMD- RS RAEB-1 RAEB-2 Karyotype Good Intermediate Poor Transfusion requirement * No Regular Risk groups: Very low score = 0 Low score = 1 Intermediate score = 2 High score = 3-4 Very high score = 5-6 * Transfusion dependency = at least one RBC transfusion every 8 weeks over 4 months Ref: Malcovati et al, JCO, 25: 3503, 2007

37 Detlef Haase: Updated risk features in MDS Prog. group Fav. Chromosome abnormalities 5q-, 12p-, 20q-, +21, -Y, 11q-, t(11q23), normal, 5q- plus one other abn Inter1 +1q, 3q21/q26 abn, +8, t(7q), +19, -21, any other single* or double abn Inter2 -X, -7/7q-, -7/7q- plus one other abn, complex = 3 abn Time to 25% AML Survival (months) Unfav. Complex > 3 abn * Most common group ie rare abnormalities

38 4q12 region Deleted region (800 kb) GSH2 FIP1L1 MORF4 PDGFRA cen tel LNX RPL21 CHIC2 KIT KDR RPCI11-120K16 RCPI11-3H20 RCPI11-24O10 Ref: Cools et al, NEJM, 2003

39 Normal metaphase Abnormal metaphase

40 Myeloma cytogenetics Chromosome abnormalities detected by both conventional cytogenetics and by fluorescence in situ hybridization (FISH) have been associated with prognosis in multiple myeloma Conventional cytogenetic analysis relies on obtaining dividing cells in culture and analysing the chromosome abnormalities Use of FISH allows abnormalities to be detected even in non-dividing cells

41 Common cytogenetic abnormalities Deletion of 13q observed in 20% by CC and 50% by FISH An IGH translocation at 14q32 with an oncogene observed in 50% patients with myeloma. Translocations resulting in dys-regulation of oncogene thought to be primary oncogenic events. IGH partner gene Chromosomal location Incidence Prognostic significance Cyclin D1 11q13 15% - Cyclin D2 12p13 <1% - Cyclin D3 6p21 2% - MAF 16q23 5% Poor MAFB 20q12 2% - MAFA 8q24.3 <1% - MMSET/FGFR3 4p16 15% Poor

42 Myeloma & FISH FISH analysis does not distinguish between the abnormal plasma cell population and other nucleated cells in the bone marrow The European Myeloma Network recommendations for FISH in myeloma 2007 state that it is not acceptable to report FISH results in myeloma without either concentrating the plasma cells or employing some means of plasma cell identification so that only these cells are scored.

43 Identifying Labelled Plasma Cells cig labelling: Classic PC morphology Antibody labelling within cytoplasm Easily distinguishable from other cells

44 Myeloma cytogenetic/fish testing Conventional cytogenetic analysis: poor prognosis abnormalities: FISH: Hypodiploidy loss of 13q t(4;14) t(14;16) Deletion of 17p (TP53)

45 Cytogenetic abnormality FISH in CLL No. cases (325) Median Survival (months) Deletion 13q 55% 133 Deletion 11q 18% 79 Trisomy 12q 16% 114 Deletion 17p* 7% 32 Normal Karyotype 18% 111 * Independent prognostic indicator (Dohner et al; Krober et al; Oscier et al 2002)

46 Monoallelic vs biallelic del(13q) Loss of one 13q14 (D13S319) signal is the commonest genetic abnormality in CLL As a sole aberration, it predicts a relatively indolent course However, it is not uncommon to find a clone with loss of both 13q14 alleles Does this carry the same prognostic significance as loss of only one allele?

47 Chena et al, European J Haematology 2008: Studied 103 CLL patients with FISH panels to detect trisomy12, deletions of ATM (11q22), 13q14 and 13q34 (D13S319 and LAMP1) and TP53 (17p13) 6/103 (6%) showed biallelic 13q14 loss as sole abnormality and 32/103 (31%) with monoallelic loss 6/6 vs 12/32 showed disease progression Treatment free survival was 28.5 months vs 49 months

48 Is it CLL or Mantle cell lymphoma?

49 MCL characterised by t(11;14)(q13;q32) t(11;14) Cyclin D1 Causes cyclin D1 upregulation (BCL1, PRAD1, CCND1)

50 FISH probe: IGH/CCND1 dual colour dual fusion translocation probe

51 double hit lymphomas Rare neoplasms characterised by highly aggressive clinical behaviour, complex karyotypes and pathological features overlapping BL, DLBCL and B-lymphoblastic lymphoma/leukaemia May have history of FL 93 cases over 3 studies: 48 B-cell lymphomas unclassifiable, 45 DLBCL*, 1 B-LBL, 1 FL commonly: stage 3-4 disease; high LDH levels; extra nodal disease; BM involvement; CNS disease Niitsu et al, Leukemia 2009 Snuderl et al, Am J Surg Pathol 2010 Johnson et al, 2009

52 MYC break apart probe Dual fusion IGH/BCL2 probe

53 Double hit lymphoma More commonly t(8;22) rather than t(8;14) or non-ig / MYC translocations plus higher number of chromosomal aberrations At 5 years, only 6 survivors and 32 died within 6 months of MYC translocation (Johnson et al) Median overall survival 4.5 months inferior to both BL and IPI matched DLBCL (Snuderl et al) Niitsu et al, Leukemia 2009 Snuderl et al, Am J Surg Pathol 2010 Johnson et al, 2009

54 Dual translocations in DLBCL Niitsu et al, Leukemia 2009

55 Acknowledgements: Staff of the VCCS Cris Batzios Karen Binnion Rebecca Bowen Melissa Curtis Pina D Achille Caroline Dobrzelak Lee Harrison Veronica Hoctor Kinjal Joshi Ruth MacKinnon Bruce Mercer Trish Michael Megan Nolan Srilakshmi Nutalapati Fran O Malley Sarah Poile Kathleen Rayeroux Anne Robertson Dora Stamatonikolos Lan Ta Meg Wall Joanne White Adrian Zordan