Do acgh analysis have a place in routine cytogenetic workup in leukemia/cancer? - A single institution experience. Cambridge, April 9 th 2013

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1 Do acgh analysis have a place in routine cytogenetic workup in leukemia/cancer? - A single institution experience. Cambridge, April 9 th 2013 Aarhus University Hospital Eigil Kjeldsen, Cancercytogenetic Lab. Centre for Cancer- and Inflammation Department of Hematology

2 Outline Cancer Cytogenetics Historical Perspective Organization of cancer cytogenetics in Denmark acgh analysis as an adjunct tool/hematology Cases acgh Summary What s comming in the future Questions

3 Good Old Times: Hematocrits - Gold Standard Plasma White cells Red cells Acute Leukemia: Death < 3 mo. Normal, Hemorrhage, IDA, Leukemia, Hemolysis, B12, P Vera

4 Modern Era: Cancer is a Genetic Disease > 95% of cases are somatic - the rest are inherited Cytogenetics in hematologic cancers is far ahead of solid tumors 1960: The first recurrent cytogenetic abnormality was the Phchromosome detected in CML 1973: The Ph-chromosome was described as t(9;22)(q34;q11) due to novel banding methods

5 Chromosomal Translocation t(9;22) in CML - Historical Perspective s 2001

6 Cancer is a Genetic Disease > 95% of cases are somatic - the rest are inherited Cytogenetics in hematologic cancers is far ahead of solid tumors 1960: The first recurrent cytogenetic abnormality was the Phchromosome detected in CML 1973: The Ph-chromosome was described as t(9;22)(q34;q11) due to novel banding methods Now >800 recurrent cancer chromosomal abnormalities are known For many of the translocations we know when they occur in hematopoiesis

7 Hematopoietic cell differentiation and chromosome abnormalities in leukemia and lymphoma ALL t(9;22) t(11q23) AML t(11q23) AML t(8;21) in(16) t(15;17) Mast cell Erythrocytes Myeloid progenitor hematopoietic stem cell Lympho-myeloid Stem cell Platelets Lymphoid Progenitor Eosinophil CML t(9;22) ALL t(12;21) t(1;19) t(8;14) Hyperdipl. B cell NHL t(8;14), t(14;18) t(11;14) T cell ALL/NHL t(14q11.2) t(7q34) Neutrophil Monocyte

8 Cancer is a Genetic Disease > 95% of cases are somatic - the rest are inherited Cytogenetics in hematologic cancers is far ahead of solid tumors 1960: The first recurrent cytogenetic abnormality was the Phchromosome detected in CML 1973: The Ph-chromosome was described as t(9;22)(q34;q11) due to novel banding methods Now >800 recurrent cancer chromosomal abnormalities are known For many of the translocations we know when they occur in hematopoiesis For many of the chromosomal abnormalities we know their impact on disease progression and response to treatment

9 % alive MRC/NCRI AML Trials: Overall Survival ages 16-59, 2550 patients, 10 years follow-up t(15;17), n=607 t(8;21), n=421 inv(16)/t(16;16), n=284 t(9;11), n=61 t(3;5), n=25 t(6;9), n=42 AML/MDS, n=343 other 11q, n=60 t(9;22), n=44-7/del(7q), n=336-5/del(5q), n=258 Inv(3)/t(3;3),n=69 Years from entry * Normal karyotypes: 38% OS Grimwade et al., Blood, April 12, 2010

10 Clinical significance of chromosome abnormalities in leukemia and MDS Diagnosis and differential diagnosis: WHO classification (2008): specific cytogenetic/molecular genetic findings, such as t(8;21), t(15;17), inv(16), t(9;11) and other 11q23/MLL, inv(3)/t(3;3), t(6;9), t(1;22). Treatment protocols according to Cytogenetic Diagnosis Monitoring response and engraftment of BMT cytogenetic complete remission (CR) and MRD Prognosis: most critical and independent indicators. favorable (55-81% cured): t(15;17), inv(16), t(8;21); intermediate (40%): t(9;11), normal karyotype; unfavorable (<5%): complex, abnl 5 and 7, inv(3), t(6;9)

11 Cancer is a Genetic Disease > 95% of cases are somatic - the rest are inherited Cytogenetics in hematologic cancers is far ahead of solid tumors 1960: The first recurrent cytogenetic abnormality was the Phchromosome detected in CML 1973: The Ph-chromosome was described as t(9;22)(q34;q11) due to novel banding methods Now >800 recurrent cancer chromosomal abnormalities are known For many of the translocations we know when they occur in hematopoiesis For many of the chromosomal abnormalities we their impact on disease progression and response to treatment Some abnormalities are difficult to detect - cryptic or submicroscopic abnormalities - FISH is an appreciated adjunct technology in these cases (e.g. 7q-)

12 Cryptic Genomic Abnormalities - FISH N=588 MDS patients 433/588 (74%) had Normal karyotype 17/433 (4%) had cryptic isolated -7/7qdetected by FISH

13 Diagnosis and Treatment of Hematological Cancers in A Multidisciplinary Work-Up 1. Diagnose a Malignant Hematologic Disease: 2. Determine Treatment: 3. Treatment Protocol: 4. Follow-up: Cytogenetics Flow cytometry 19 % 41 % 41 % Pathology? Microarray 9 % 9 % 9 % 73 % PCR methods 70 % 10 % 10 % 10 % Acute Leukemia in Childhood: 5 years survival >80% 1. Chromosomal banding is Gold Standard for cytogenetic leukemia work-up 2. FISH/PCR are adjunct methods for specific genetic abnormalities 3. acgh/snp tools for research alone!?

14 5 Cancercytogenetic Laboratories in DK AUH: 1,802 mio in pop OUH/Vejle: 1,116 mio in pop Holstebro Silkeborg Århus Randers RH/JFK: 2,431 mio in pop Samples/yr/lab

15 How do we practice Cytogenetics, FISH and acgh in malignant hematology and in solid tumors? Primarily Leukemia and Preleukemia patients All NOPHO protocol patients Selected AML17 patients All NOPHO protocol patients incl. young adults: 1. G-banding 2. Target specific FISH 3. acgh analysis 180K Cytochip Cancer BlueFuse Multi v. 3.6 and Nexus v. 6.1 Report of all 1-3: on average 4-5 working days Unusual cases of AML, unexplained abnormal hematology, and others > acgh as adjunct tool Research dnaml, saml, Renal cancer, cell lines We have analyzed >450 arrays: Apprx K-CytoChipCancer ( >) Apprx. 350 CytoChip BAC arrays ( > )

16 Cancer Cytogenetics Chromosomes: In leukemia cells morphology is often poor - few short and fuzzy bands, whereas in normal cells nice bands. Analysis: Heterogeneous populations (normal, abnormal clones). Precise hematopathological diagnosis is important for targeted detection of recurring chromosome abnormalities in specific subtypes.

17 Cryptic der(5)t(4;5)(q26;q21.1) in T-ALL - 1 Partial Karyotype of chr. 4 and 5 A B C Normal individual E F T-ALL patient D Nor G q26 q21.1 Kjeldsen & Rough, Mol. Cytogenetics (2012)

18 Novel cryptic der(5)t(4;5) in T-ALL - 2 A B C E F D G q26 q21.1 Mitelman Database search for t(4;5) Kjeldsen & Roug, Mol. Cytogenetics (2012)

19 Aneuploidy case yr-old male Pre B-ALL acgh analysis showed: Clearly, aneuploid but is it hyperdiploidy, hypodiploidy or a mix?

20 Aneuploidy case 2: G-Banding G-banding showed: 34,X,-Y,-2,-3,-4,-7,-9,del(10)(q23),-12,-13, -15,-16,-17,-20 [8] 46,XY [14] 66<3n>,XX,-Y,+1,-2,-3,-4,-9,-10,-13,-17,-20, +22,+mar [3] ifish with BCR,ABL probe: 2R2G [51]/1R2G [37]/2R4G [12]

21 Aneuploidy case 2: acgh Diploid Correction No Correction for Diploid Regions: Correction with Diploid Regions: arr[hg18] -2,-3,-4,-7,-9,del(10)(q24.1q26.3),-12,-13,-15,-16,-17,-20,-Y

22 Renal Cancer Project: acgh findings and Treatment Response In Renal Cancer Project: Reference DNA is purified from the patients blood => The profiles are more smooth and have reduced copy number varinats In Leukemias: Reference DNA is Promega Male or Female pooled DNA

23 acgh as a tool in MDS Slovak et al. J Mol Cytogenet (2010) N=30 MDS patients: comparison acgh, FISH and G_banding Unexpected result: Dup-Del-Dup Cryptic aberrations found by acgh (BAC array) in 14/30 (47%) acgh analysis improve chromosomal findings

24 Prognostic Scoring Systems in Hematology - MDS as an example MDS patient with del(20q) progressed to AML Retrospective acgh analysis revealed cryptic del(5q) Prognostic scoring based on G-banding alone: 1. Good prognosis because of del(20q) 1. If G-banding revealed del(5q) and del(20q) the prognosis would be Intermediate 3. Because del(5q) only convincingly was detected by acgh analysis and confirmed by a specific BAC probe: Prognosis was still designated as Good

25 Algorithm for Cyogenetic Testing of del(5q) in MDS?? acgh/snp part of suggested algorithm

26 Summaries

27 Potentials and Problems of acgh/snp in Cancer High resolution - No dividing cells Detect copy number alteration and for SNP UPD Provide new insights of the genetic mechanisms of leukemia/mds No balanced translocations, inversions or sequence mutations Low sensitivity, 20-30% abnormal cells minimal Mosacisms and clonal evolution? Primary or secondary changes? Clinical significance of genomic aberrations? Normal variants Type of Reference DNA How to define recurrent abnormalities from acgh Minimal common regions after analysis of large cohorts then?fish/pcr acgh/snp also generates data from regions with no/very little bioinformatic information what to report and what not to report? Survival, prognosis, subclassification, risk grouping and treatment?

28 Comparison of cytogenetics, FISH and acgh Techniques Cytogenetics FISH acgh/snp Resolution Sensitivity Unbalanced cell division + balanced lesions + + -/+ multiple clones + /+ Screening for unknown lesions Normal variants -/

29 Conclusions We find that acgh analysis is an important adjunct tool in the cytogenetic work-up of leukemia - and in cancer/renal cancer We don t recommend that acgh/snp analysis should be done on all hematological patients up front in many cases acgh confirms Conventional Cytogenetics We recommend that it should be part of the cytogenetic toolbox to be used in certain subsets of patients - perhaps like 24-color karyotyping Its an expensive tool (running cost/investment) and a highly skilled tool core facility!? As long as acgh/snp isn t part of diagnostic algorithms or treatment protocols it ll be regarded more as a research tool than a routine diagnostic tool. Who should pay for the service in Germany, USA,?UK it s often insurance companies

30 CML t(9;22)(q34;q11) to Modern Era - Meet the Expert ASH Dec

31 Can cytogenetics, FISH and acgh survive in the next modern genomic era? Next-generation sequencing Yes!? For cytogenetics and FISH- valuable single cell analysis being able to identify different clonal abnormalities. For acgh analysis: advantage is speed relative to NGS

32 Questions?