The role of cytogenomics in the diagnostic work-up of Chronic Lymphocytic Leukaemia

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1 The role of cytogenomics in the diagnostic work-up of Chronic Lymphocytic Leukaemia Adrian Zordan, Meaghan Wall, Ruth MacKinnon, Pina D Achille & Lynda Campbell Victorian Cancer Cytogenetics Service (VCCS)

2 Chronic Lymphocytic Leukaemia (CLL) CLL accounts for approximately 25% of all leukaemia and is the most common lymphoid malignancy in Western countries It is usually an indolent disease characterised by an accumulation of CD5+ abnormal B lymphocytes Median age at diagnosis: 72 years

3 Genetic Prognostic Indicators Conventional cytogenetics: Translocations appear to indicate a poor prognosis, particularly: t(14;19), MYC translocations and possibly t(14;18) Complexity (defined as 3 or 5 chromosome aberrations associated with a poor prognosis FISH: del(13q) (DLEU2) as a sole abnormality an indicator of relatively indolent disease (but larger deletions encompassing RB1 have less favourable outcome) Trisomy 12 a marker of intermediate prognosis; often associated with atypical morphology del(11q) (ATM) associated with a poor prognosis del(17p) (TP53) associated with a very poor prognosis

4 The VCCS CLL Microarray Pilot Study FISH based Prognostic markers for CLL make it a prime candidate for single-nucleotide polymorphism microarray (asnp). Reported abnormality rates: CC 51% to 98% abnormality detection rate (~60% in our experience) FISH testing up to 80% detection rate. asnp up to 100% Can whole genome microarray using asnp be substituted for current techniques?

5 The VCCS CLL Microarray Pilot Study 29 CLL samples randomly selected CC, FISH & asnp run in parallel CC and FISH performed on cultured samples asnp applied DNA from uncultured lymphocytes 2 asnp platforms trialled: Illumina CytoSNP12 (24 Samples) Affymetrix CytoScan 750K array (5 Samples)

6 Overall abnormality detection rate CC CC only performed on 25/29 samples 24/25 samples cultured & analysed successfully Abnormalities detected in 16/24 (67%) FISH All 29 samples FISHed with CLL probe panel (ATM/TP53 DC, D12Z3 SC, DLEU2/LAMP1 DC) Abnormalities detected in 24/29 (83%) samples asnp All 29 samples processed with asnp Abnormalities detected in 25/29 (86%) samples

7 Overview of alterations detected Conventional Cytogenetics FISH 6* asnp * Balanced translocations detected only by CC

8 % of total samples Detection of known CLL abnormalities by CC vs. FISH vs. asnp CC FISH asnp 7 0 del(13q) del(13q) as sole abn without ATM or TP53 deletion ATM deletion TP53 deletion NB Total number of samples = 29. CC not available for 5/29.

9 Abn. Detection rate Investigation of Abnormality detection rates Combined abnormality detection rate: Including abnormalities of uncertain clinical significance = 26/29 (90%) samples Restricted to known recurrent CLL alterations = 25/29 (86% ) samples Combined asnp FISH CC

10 Investigation of Abnormality detection rates Total abnormalities detected per sample Number of abnormalities detected per sample CC FISH asnp

11 Investigation of discordant results Discordant results between FISH and asnp were observed in 3/29 (10%) samples: D13S319x1,LAMP1x2 [22/200] No deleted chromosome 13 material detected using CytoSNP12 D13S319x1,LAMP1x2 [21/200] No deleted chromosome 13 material detected using CytoScan 750K ATMx1,TP53x2 [55/200] Despite passing recommended QC levels, not analysable due to noisy data.

12 Discordant results FISH vs. asnp asnp results for chromosome 13, where corresponding FISH shows ~10% abnormal nuclei

13 Discordant results asnp vs. FISH RB1 & DLEU2 FISH PROBES 100kb 905kb 2038kb del(13) FISH with RB1 /DLEU2 /LAMP1 TC probe.

14 Discordant results copy neutral LOH 11q13.1q25(65,670, ,442,936)x2 hmz 9q13q34.3(70,224, ,164,585)x2 hmz

15 Complex abnormalities revealed by asnp 3cth 11cth del(2) del(13)

16 Conclusion the role of cytogenomics in CLL asnp is a valuable tool for diagnostic study of CLL CC and FISH cannot yet be substituted by asnp as: Independent prognostic value of many of the abnormalities detected by asnp requires further investigation One instance of a poor prognosis abnormality detected by FISH only These techniques are complementary: CC provides information regarding structural events FISH has been shown to offer the highest sensitivity asnp has the greatest resolution Further studies required to define the prognostic value of novel asnp abnormalities CC and FISH to remain the techniques of choice with asnp as a valuable complementary tool.

17 Future directions and suggestions More targeted cancer asnp chips required Greater SNP probe coverage in key genes/regions Cell purification suggested for diagnostic application of asnp. asnp can replace FISH once the sensitivity and economics have been addressed

18 Acknowledgements Meaghan Wall, Ruth MacKinnon, Pina D Achille and Lynda Campbell for their direct involvement in the pilot study Affymetrix & Illumina for providing asnp chips for evaluation Darren Jardine & Molecular Diagnostics Dept at St. Vincent s Hospital - facilitating the DNA extraction of our diagnostic samples Staff at VCCS