Cytogenetics Update Lynda J Campbell lynda.campbell@svhm.org.au Ph - PowerPoint PPT Presentation

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

Ph Nowell and Hungerford, 1960

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

Acute lymphoblastic leukaemia

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

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

HEH karyogram

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 CEP4, 10, 17 ETV6/RUNX1

t(4;11)(q21;q23) • 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

Ph+ ALL • 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

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)

Multiple copies of RUNX1 clustered on a marker chromosome

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.

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 Improved outcome Ph translocation High hyperdiploidy t(4;11)(q21;q23) Deletion of 9p Burkitt translocation * Complex karyotype (≥ 5) * Low hypodiploidy/triploidy * * Independent of sex, age, WCC and T-cell Moorman et al, Blood 2007

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

Cytogenetically Normal AML (CN-AML) Acute Myeloid Leukaemia with t(8;21); RUNX1- Figure 1. Pie chart illustrating RUNX1T1 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: 453-474, 2010

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

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 RARA with PML/RARA probe PRKAR1A BAC probe

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

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

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

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

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

Overall survival curve for patients with 11q23/ MLL - rearranged 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. 2489-2496

Recent case: 23 year man presented with AML 10 11 p13 (AF10) q14 (CALM) q23 (MLL)

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)

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…

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

Prognosis categories in AML MRC % SWOG/ECOG % Good Inv(16)/t(16;16) ; 21 Inv(16)/t(16;16) ; 20% t(15;17), t(8;21) +/- % t(15;17)+/- other abn; other abn; t(8;21) without del(9q) or Cx Intermediate Normal, 11q23 abn, 62 Normal, +8, +6, -Y, 46% +8, del(9q), del(7q), % del(12p) +21, +22, all others Poor Del(5q)/-5, -7, abn 17 Del(5q)/-5, -7/del(7q), 30% 3q, and Complex (≥5 % abn 3q, 9q, 11q, 20q, unrelated abn) 21q, 17p, t(6;9), t(9;22) and Complex (≥3 t(6;9)*, t(9;22)* unrelated abn) Unknown N/A All other abn 4% Ref: Slovak et al, Blood, 96: 4075, 2000; Grimwade et al, Blood, 92: 2322, 1998

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); Normal Karyotype: t(9;11)(p22;q23); Inv(3q) or t(3;3); RUNX1-RUNX1T1 Mutated NPM1+ MLLt3-MLL RPN1-EVI1 Inv(16) or t(16;16) FLT3 ITD Cytogenetic t(6;9)(p23;q34); CBFB-MYH11 Wild type NPM1+ abnormalities not DEK-NUP214 Normal karyotype: FLT3 ITD classified as t(v;11)(v;q23); MLL Mutated NPM1 (no Wildtype NPM1 + favourable or -5 or del(5q) FLT3) no FLT3 ITD adverse -7 Mutated CEBPA Abnormality of 17p Complex karyotype: ≥3 abnormalities Döhner et al : Recommendations on behalf of the European LeukemiaNet, Blood 2010

Acquired uniparental disomy in CN-AML Bollinger et al , Leukemia (2010) 24, 438 – 449

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

IPSS for MDS: Survival and AML evolution Score Value Prognostic 0 0.5 1.0 1.5 2.0 variable BM blasts (%) <5 5-10 - 11-20 21-30 Karyotype # Good Intermediate Poor Cytopenias* 0/1 2/3 # Good: normal, -Y, del(5q), del(20q) complex (  3) or chromosome 7 abn. Poor: Intermediate: other abnormalities * Cytopenias: Hb <100g/L, Neutrophils <1.8x109/L, Platelets <100x109/L Ref: Greenberg et al, Blood 89: 2079-2088, 1997

WPSS for MDS Variable 0 1 2 3 WHO category RA, RARS, 5q- RCMD, RCMD- RAEB-1 RAEB-2 RS Karyotype Good Intermediate Poor − Transfusion No Regular − − requirement * 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

Detlef Haase: Updated risk features in MDS Prog. Chromosome Time to Survival group abnormalities 25% AML (months) Fav. 5q-, 12p-, 20q-, +21, -Y, 71.9 51 11q-, t(11q23), normal, 5q- plus one other abn Inter1 +1q, 3q21/q26 abn, +8, 16 29 t(7q), +19, -21, any other single* or double abn Inter2 -X, -7/7q-, -7/7q- plus one 6 15.6 other abn, complex = 3 abn Unfav. Complex > 3 abn 2.8 5.9 * Most common group – ie rare abnormalities

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

Abnormal Normal metaphase metaphase