HD-SNP Microarray Analysis of the Study 9 High Risk ALL Patients - Increased Yield of Important Prognostic Information Nadine K Berry 1,2,4, Rodney J. Scott 1,4, Rosemary Sutton 5, Toby N Trahair 5, Philip Rowlings 2,3, Anoop K Enjeti 2,3 1. Department of Molecular Medicine, NSW Health Pathology, Newcastle 2. Department of Haematology, Calvary Mater Hospital, Newcastle 3. School of Medicine and Public Health, University of Newcastle, Newcastle 4. School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle 5. Children s Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Randwick
Pilot Study Australian patients enrolled on AIEOP-BFM ALL 2009 All in the high risk group (15% of patients) B-ALL (n=15) 9/15 = Male 6/15 = Female 2.6yrs 16.5yrs T-ALL (n=7) 7 = Male 3.1yrs 16.5yrs Cohort n=22
HD-SNP Microarray Affymetrix HD CytoScan microarray analysed in ChAS Analysis protocol with guidance from the CCRI, Vienna Cyto regions Gain = 25 markers Loss = 20 markers Genome wide Gain = 50 markers / 50kb Loss = 25 markers / 25kb LOH 3Mb with special consideration for regions extending to the telomere
AIEOP-BFM ALL 2017 (Proposed Genomic Risk) High Risk KMT2A-rearranged? iamp21 Hypodiploidy t(17;19) IKAROS Plus IKZF1deletion PLUS P2RY8-CRLF2 deletion or CDKN2A /B deletion or PAX5 deletion Non CR and high MRD are separate high risk factors Note: High Risk BCR-ABL1 = t(9;22) Excluded, treated on TKI protocol Non-High Risk IKZF1del/+ ERG deletion IKZF1del only
Deletions of IKZF1 MLPA - IKZF1(7p12.2) IKZF1 PCR IKZF1 Exon 01 IKZF1 Exon 02 IKZF1 Exon 03 IKZF1 Exon 04 IKZF1 Exon 05 IKZF1 Exon 06 IKZF1 Exon 07 IKZF1 Exon 08 HD SNP scikzf1d4-7 Normal (1.01) Normal (1.01) Normal (1.02) Normal (1.07) Normal (0.97) Normal (0.9) Normal (1.09) Normal (0.86) Nil Change IKZF1D4-7 Normal (0.95) Normal (0.9) Normal (0.97) LOH (0.48) LOH (0.57) LOH (0.58) LOH (0.48) Normal (1.11) Nil Normal (0.91) Normal (0.86) Normal (0.76) Normal (0.71) Ambiguous (0.65) Normal (0.93) Normal (0.96) Ambiguous (0.64) Nil Ambiguous (0.63) Ambiguous (0.64) LOH (0.5) LOH (0.5) LOH (0.54) LOH (0.58) Ambiguous (0.62) LOH (0.46) IKZF1D4-7 Normal (0.99) Normal (0.96) Normal (1.05) LOH (0.56) LOH (0.56) LOH (0.52) LOH (0.55) Normal (1.02) Nil Normal (0.91) Normal (0.89) Normal (0.89) Normal (0.78) Normal (0.8) Normal (0.8) Normal (0.83) Normal (0.86) IKZF1D4-7 Normal (1.2) Normal (1.17) Normal (0.88) LOH (0.55) LOH (0.45) LOH (0.56) Ambiguous (0.68) Normal (0.71) Loss (e4-e7) WC Loss (~95%) WA Loss (~90%) Loss (e4-e7) WA Loss (~38%) Loss (e4-e7)
P2RY8-CRLF2 fusion Not to Scale
MLPA Xp22.33-PAR region HD-SNP 36kb Deletion (32 Markers) Array ID CRLF2 Exon 04 CSF2RA Exon 10 IL3RA Exon 01 P2RY8 Exon 02 CSF2RA IL3RA B1 Normal (1.12) Normal (0.96) Normal (0.88) Normal (1.1) e11-14 e1-2 e11-14 e1-2
Patient B1 (? CSF2RA-IL3RA fusion)
Patient B1 (? CSF2RA-IL3RA fusion) Female 10years 8months Pre-B WCC 10.8x10 9 /L NCI-Rome Risk = High 46,XX[34] Normal FISH (D4Z1,D10Z1,D17Z1)x2[200] (ABL1,BCR)x2[200] (KMT2Ax2)[200] (ETV6,RUNX1)x2[200] (TCF3x2)[200]
Sutton. R (CCIA) *
B1 Microarray Results CN Type Chr Size (kbp) Markers Gene Count OMIM Genes Cyto Regions Call Interpretation 1 Loss X 38.09 32 2 CSF2RA, IL3RA IL3RA Pathogenic del(x)(p22.33) CN=1 (3` loss CSF2RA - exons 14-17)(5` loss IL3RA exons 1-2) FUSION GENE? CSF2RA-IL3RA 1 Loss 9 108.01 168 1 PAX5 PAX5 Pathogenic del(9)(p13.2) CN=1 (Intragenic loss PAX5 - exons 2-6) 3 Gain 7 601.67 656 3 GNA12, CARD11, SDK1 Likely Pathogenic dup(7)(p22.3p22.2) CN=3 No onco genes (5` gain GNA12 - exons 1-2) (5` gain SDK1 - exon 1) 4 Gain 17 43.28 88 1 HLF HLF Likely Pathogenic dup(17)(q22) CN=4 (3` gain HLF - exons 3-4) -?HLF rearrangement. Check TCF3
B1 B1 Expression of CRLF2 in Diagnosis BM MNC in High Risk ALL (blue) or Medium Risk ALL with IGH-CRLF2 (orange) or P2RY8-CRLF2 (red) Sutton. R (CCIA)
B1 B1 Expression of IL3RA in Diagnosis BM MNC in High Risk ALL (blue) or Medium Risk ALL with IGH-CRLF2 (orange) or P2RY8-CRLF2 (red) Sutton. R (CCIA)
Patient A6 (IL3RA e2 amplification)
A6 A6 Expression of CRLF2 in Diagnosis BM MNC in High Risk ALL (blue) or Medium Risk ALL with IGH-CRLF2 (orange) or P2RY8-CRLF2 (red) Sutton. R (CCIA)
A6 A6 Expression of IL3RA in Diagnosis BM MNC in High Risk ALL (blue) or Medium Risk ALL with IGH-CRLF2 (orange) or P2RY8-CRLF2 (red) Sutton. R (CCIA)
HR Features (B-ALL) Array ID HR FISH HR Karyotype PPR (PB) HR Flow HR PCR MRD (24hrs) (7days) (Day 8) (Day 15) TP2 (Day79) A1 x x A2 x A3 x B2 x B11 x x B9 x B6 x B8 x A5 x x A11 x x A6 x B4 x B1 x A9 x A10 x x
HR Features (B-ALL) Array ID A1 HR FISH (24hrs) HD-SNP Microarray (7days) IKZF1 / PAX5 / CDKN2A / B HR Karyotype (7days) PPR (PB) (Day 8) HR Flow (Day 15) HR PCR MRD TP1 (Day 33) HR PCR MRD TP2 (Day79) x x x A2 x x A3 B2 B11 IKZF1 / CDKN2A x x x B9 IKZF1 / iamp21 x B6 B8 A5 IKZF1 x x x A11 IKZF1 x x x A6? IL3RA e2-amp x x B4 B1 CSF2RA-IL3RA x x A9 Hypo Hypo / IKZF1 A10 KMT2A+ IKZF1 t(4;11) x x x x x
HR Features (T-ALL) Array ID A4 HR FISH (24hrs) HR Karyotype (7days) PPR (PB) (Day 8) X HR Flow (Day 15) B7 X X HR PCR MRD TP2 (Day79) A12 X X A7 X X B3 X X A8 X X B5 X X
Acknowledgments Children s Cancer Institute Prof Rosemary Sutton Nicola Venn Tamara Law Benjamin Schreiber Walter Muskovic Anu Dissanayake Louise Doculara Jodie Giles Dr Carol Wadham Dr Toby Trahair Prof Murray Norris Participating Laboratories SA Pathology - Sarah Moore Children s Hospital Westmead SEALS - Pauline Dalzell Pathology North (Newcastle) Trial Hospital Site Coordinators Dr Draga Barbaric Dr Luciano Dalla Pozzo Dr Tom Revesz Dr Frank Alvaro Children s Cancer Research Institute (CCRI), Austria Oskar Haas Karin Nebral Sabine Strehl Andrea Inthal Funding Support NHMRC grant APP1009087 Norris NHMRC grant APP1057746 Sutton Tour de Cure Haber Pathology North Charitable Trust
Clinical significance of distinct types of IKZF1 deletions. Different types of IKZF1 deletions can be observed, but whether they equally affect prognosis is still an open question. We subdivided IKZF1del cases into three groups: whole-gene deletions resulting in haploinsufficiency, including the loss of chromosome 7p (n = 66, 37%), intragenic deletion of exons 4 7, producing dominant negative isoforms (Δ4 7, n = 62, 35%) and rare intragenic deletions (n = 51, 28%). There was no significant difference (P = 0.72) in EFS according to the type of deletion
Rare variant types with the most unfavorable event-free survival were DEL 2 7 (P=0.03), DEL 2 8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4 7 and DEL 1 8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.
Blood 2010 116:4874-4884; doi: https://doi.org/10.1182/blood-2009-08-239681 frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA High CRLF2 expression
Deletion of CSF2RA and IL3RA with normal CRLF2, an aberration predicted to cause CRLF2 overexpression, 12 was detected in 4.3% of cases.
P2RY8-CRLF2 fusion http://atlasgeneticsoncology.org/anomalies/delxp22pr2y8-crlf2id1599.html
Normal=1 on the graph not 0. Expression levels were measured by RQ-RT-PCR assays. Levels were normalised using a house keeping gene (RALGAP1 a house keeping gene) on same sample/run and then by level of CRLF2 expression in normal paediatric bone marrow (same run). (For your information - but not to write on a slide - this was bone marrow from an identical twin of another patient with ALL collected to make sure he did not have ALL too - parents agreed that sample could be used for research).