Emma Huxley. Principal Clinical Scientist West Midlands Regional Genetics Laboratory

Transcription

1 Genetic Analysis Using a High Density SNP Array in Myelodysplastic Syndrome: Clinical Utility and Comparative Analysis Study Compared to Metaphase Chromosome Analysis. Emma Huxley Principal Clinical Scientist West Midlands Regional Genetics Laboratory

2 Disease of the elderly? MDS related clone Short survival Myelodysplastic syndrome A clonal haemopoietic stem cell disorder Diagnosis is challenging Bone marrow required 50% cytogenetic clonal detection rate (WHO, 2008) G-band analysis is mandatory (ELN, IPSS-R)

3 The MDS SNP array project Funded by Affymetrix for 2 years Demonstrate clinical utility of Cytoscan HD array Complementary to G-band analysis Presentation MDS cases or continued suspicion of MDS (BM and paired PB)

4 Why use SNP arrays? Cytogenetic changes in MDS are largely copy number (CN) changes SNP arrays also allow for detection of regions of copy neutral loss of heterozygosity (CN-LOH) Resolution is beyond that of G-band chromosome analysis kb vs Mb No need for metaphases, just DNA Potential for blood to be analysed instead of marrow, meaning samples could be obtained from patients too frail for bone marrow aspirate Potential to reduce failure rates

5 Affymetrix CytoScan HD Array High density array with over 2.6 million markers 750,000 genotype-able SNPs 1.9 million non-polymorphic probes Can reliably detect changes down to 25 kb

6 SNP arrays Genomic backbone probes SNP probes A or B variant and ratio of each Smooth signal Copy number state Regions of loss of heterozygosity Genes G-banded chromosome

7 Copy neutral loss of heterozygosity (CN-LOH) Duplication of the maternal or paternal chromosome or chromosomal region and concurrent loss of the other alleles Germline or acquired Whole chromosome or segmental Large, telomeric CN-LOH suggests acquired Can result in doubling up of mutated genes or complete loss of genes if deleted CN-LOH is seen in MDS

8 Results Increased number of genetic aberrations (CN changes and CN- LOH) detected by SNP array 53/150 (35%) by metaphase analysis and 82/150 (55%) by SNP array analysis

9 33 cases with a normal karyotype by G-band analysis showed genetic aberrations by SNP array 4 cases with an abnormal karyotype had a normal SNP array 2 low level abnormal clones (~4%) 2 balanced rearrangements inv(5)(p15q3) inv(11)(q21q23) Detection of abnormal clones with CN changes at sensitivity as low as between 6 and 20%

10 Detection of CN-LOH regions, which contain bi-allelic gene mutations, in 20/150 (13%) cases, including chromosomal regions 1p (MPL, NRAS), 4q (TET2), 7q (CUX1), 11q (CBL), and 17p(TP53) Regions of CN-LOH detected by SNP array 7q 11q

11 Detection of deletions at the single gene level, including CUX1, TET2 and RUNX1 RUNX1 CUX1 Detection of copy number changes within genes, including duplication within KMT2A Consistent with partial tandem duplication

12 Case Study 1 57 year old female?mds, thrombocytopenia, fatigue, bruising G-band chromosome analysis: 48,XX,+19,+21[4]/ 49,XX,+8,+19,+21[6]

13 7 10% 8 60% 19 84% 21 80%

14 Final karyotype: 48,XX,+19,+21[16]/ 49,XX,+8,+19,+21[39]/ 47,XX,-7,+19,+21[5] IPSS-R cytogenetic risk group changed from poor to very poor

15 Case study 2 75year old male Confirmed MDS RARS G-band chromosome analysis: 46,XY[20] 49.6 Mb CN-LOH 11q14.1qter Recurrent finding in myeloid neoplasia CMML, MDS/MPN and atypical CML Associated with CBL mutation Mutation analysis on-going

16 83 year old female?mds Case Study 3 Anaemia with thrombocytopaenia Unable to obtain marrow PB sent specifically for SNP array analysis Two deletions of 5q: ~8Mb 5q14.3->5q15 ~56Mb 5q21.1->5q33.3

17 Case Study 4 73 year old female MDS,?progression to AML G-band chromosome analysis: 47,XX,+21[4]/ 46,XX[6] Whole gain of chromosome 21

18 12 Kb gain of 11q23.3

19 Support findings of G-band analysis Trisomy 21 In addition, Detection of partial duplication within KMT2A (MLL) gene at 11q23.3 Likely to represent partial tandem duplication (PTD) of KMT2A Recurrent finding in myeloid neoplasia

20 Case Study 5 14 year old male?hypoplastic MDS,?aplastic anaemia G-band chromosome analysis: 46,XY[20]

21 2 regions of CN-LOH of 6p: 6pter->6p pter -> 6p12.3

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23 CN LOH 6p including HLA gene complex region is recurrent finding in AA Typically see multiple clones (two observed in this patient) Process not driven by malignancy May be mechanism of immune escape in this disease SNP array able to distinguish between MDS and AA

24 Case Study 6 61 year old female Known CLL. Now confirmed secondary MDS G-band chromosome analysis: 45,XX,add(5)(q1),-7[4]/ 46,XX,add(5)(q1),-7,+mar[5]/ 46,XX[1]

25 ~28 Mb deletion of 6q14.3q22.1 ~36 Mb deletion of 13q14.12q31.1

26 ~30 Mb CN-LOH of 20q11.22 to 20qtel Deletions of 6q and 13q and CN-LOH of 20q are recurrent findings in CLL. No evidence of abnormalities observed by metaphase analysis (del(5q) and -7) Presence of two concurrent diseases: CLL and MDS

27 Conclusions so far. Advantages of using SNP array: Detect abnormalities below the resolution of G-banding Detect abnormal clones with CN changes at sensitivities as low as between 5 20%, dependent on aberration size Detect regions of CN-LOH Help define/refine breakpoints Ability to utilise PB instead of BM as no dividing cells are required, thereby allowing genetic analysis in patients too frail or otherwise unsuitable for BM Ability, when performed as a complementary tool to metaphase analysis, to detect subclones with different CN changes Challenges of using SNP array: Cannot detect genuinely balanced rearrangements Presence of concurrent neoplasia Cannot determine tetraploidy and certain amplifications

28 Use blood instead of bone marrow?% patients with a clone Improve diagnostic yield Improve patient care, QOL and outcome Expected benefits Improve turn around times Direct the treatment Increase confidence in diagnosis and risk stratification

29 Acknowledgements West Midlands Regional Genetics Laboratory: Sally Jeffries Nicola Trim Laura Ford Paula Wojtowicz Jane Soden Judith Caddick Mike Griffiths Affymetrix