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Genetic Biomarkers Revealed: Unraveling the Complexities of Cancer Genomes in Blood Malignancies

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Webinar Series Science

Sponsored by:

Participating Experts:

Detlef Haase, M.D., Ph.D. University of Göttingen Germany

Stuart Schwartz, Ph.D. Labcorp Research Triangle Park, NC

Brought to you by the Science/AAAS Custom Publishing Office


30 January 2013


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Cytogenetic Changes in MDS: – Genetic Biomarkers for Prognosis, Genetic Evolution and Personalized

Treatment Decisions

Detlef Haase and Christina Ganster Department of Hematology and Oncology

Comprehensive Cancer Center University Medicine Göttingen, Germany

Genetic Biomarkers revealed: Unraveling the Complexities of Cancer Genomes in Blood Malignancies, January 30, 2013

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• Heterogenous group of clonal hematopoetic stem cell diseases

• Impaired differentiation, growth and function of bone marrow progenitors and peripheral blood cells with peripheral cytopenias

• Often combined defects of two or all three cell lineages

• ~30% develop acute myeloid leukemias (AML)

• Increased risk for MDS-related death (bleeding complications, infections)

• Clonal genetic abnormalities (cytogenetic and molecular) can be found in almost every patient, however are very heterogeneous

• Genetically highly dynamic with frequent genetic evolution over time

• Genetic analyses gain increasing influence for individualized treatment decisions

Definition of Myelodysplastic Syndromes (MDS)

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• ~ 50% of pts. display clonal chromosome abnormalities by banding analysis 1

• ~ 50% of pts. show clonal chromosome abnorm. by SNP-Analyis2,3

• 50 to 70% of pts. have point mutations 4,5

Genetic Background of MDS

1Haase et al., Blood, 2007; 2Mohamedali et al., Blood 2007; Tiu et al., Blood, 2011 3Bejar et al., New Engl J Med, 2011; 4Bejar et al., JCO, 2012

49% 25%

26%

Cytogenetics SNP point mutation?

The aim: 100% informative cases by combining the methods

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Genetic Background of MDS Short Nucleoitide Polymorphisms: In 119 low-risk MDs pts. 46% showed UPD (A), 10% deletions (B) and 8% amplifications (C)

2Mohamedali et al., Blood 2007

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Genetic Background of MDS

Short Nucleoitide Polymorphisms:

Mohamedali et al., Blood 2007;110:3365-3373

„ SNP microarray analysis in low-risk MDS patients reveals hitherto unrecognized UPD and CN changes that may allow stratification of these patients for early therapeutic interventions“

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• Normal/non informative MC: 54% clonal abnormalities by SNP

• MC abnormal: 62% additional SNP-changes

Genetic Background of MDS Short Nucleoitide Polymorphisms: Comparative analysis of metaphase cytogenetics (MC) and SNP-Analysis in 430 pts. (MDS=250, MDS/MPN=95, Sec. AML=85):

Tiu et al., Blood 2011;117(17):4552-60

49%

44%

7%

MC normal aberrant no information

26%

74%

MC + SNP normal aberrant

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Combined (MC + SNP) prognostic scoring system

Tiu et al., Blood 2011;117(17):4552-60

• Cytogenetic risk groups according to old IPSS were considered

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Genetic Evolution of MDS

AML

?

complex Trisomy 8

5q-

5q-, komplex

Normal karyotype

Bone marrow blasts

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Patterns of Evolution in MDS, n= 46 Patienten (according to Tricot et al., 1984), own modification

P53 ?

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Genetic Evolution of MDS

AML

Bone marrow blasts

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Patterns of Evolution in MDS, n= 46 Patienten (according to Tricot et al., 1984), own modification

?

CD34 FISH

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CD34-FISH

• Circulating CD34+ cells show the same chromosomal aberrations as bone marrow cells (Haase et al., Blood, 1995, Leukemia, 1997)

• Increased number of circulating CD34+ progenitor cells in peripheral blood in MDS patients (Vehmeyer et al. Leuk. Res., 2001)

• Circulating CD34+ cells can be enriched from peripheral blood by immunomagnetic cell sorting (MACS®)

• Most chromosomal aberrations found by banding procedures can also be identified by FISH

• FISH analyses can be performed on enriched circulating CD34+ cells

Background

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CD34-FISH The Method

• 20-30 ml peripheral blood

• Immunomagnetic Cell Sorting of CD34+ cells (MACS®)

• 80 000 – 400 000 CD34+ cells/sample

• FISH analyses

Braulke et al., Leuk Res 2010

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CD34-FISH The Pilot Study

Braulke et al., Leuk Res 2010

Analysing circulating CD34+ cells by FISH…

• is a reliable method to survey the size of an aberrant clone in peripheral blood

• works in high-risk as well as in low-risk MDS

• is representative for the clonal situation in the bone marrow

• allows a very frequent monitoring during therapy

• may reduce the need for repeated bone marrow biopsies

• helps in all cases where a bone marrow biopsy is not possible or unsuccessful

• n=27 pts. with MDS • n=16 pts. with higher risk MDS and at least 4 cycles of 5-Aza

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CD34-FISH

Super-panel

Initial screening

M2

M4

M6

M8

M10 M 12 Mo

24 M 21

M18

M15

Superpanel Standardpanel Standardpanel

1. year follow-up 2. year follow-up

Super-panel

M30

M27

M33

Mo 36

3. year follow-up

Super-panel

Standardpanel

Superpanel: 12 probes: D7/CEP7, EGR1/D5, CEP8, CEP X/Y, D20, TP53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p/1q, CSF1R

Standardpanel: 7 probes: EGR1/D5, D7/CEP7, CEP8, TP53, D20, TEL/AML1, CEP X/Y

• Bone marrow biopsies every 6-12 months (recommendation)

• Peripheral blood counts once a month

• Full blood counts at time points of FISH analyses

Design of the Study

Multicentric German prospective CD34 FISH Study

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CD34-FISH Interim Study Results:

Patients n=389

• Participating Centers n=18 • Male: female 1.15: 1.0 • Median age 70.5 years (40-90) • Median time of follow-up 8.2 Months (1 – 36) • LeMon5-Study n=110 • AZALE-Study n=25

38%

72%

Cytogenetics by CD34 FISH from pB (all pts.)

normal

aberrant

61% 18%

8% 9%

2 1

Frequencies of aberrations

Pat. mit 1 Anomalie

Pat. mit 2 Anomalien Pat. mit 3 Anomalien Pat. mit 4 Anomalien Pat. mit 5 Anomalien

45% 55%

Cytogenetics by CD34 FISH from pB (without AZALE/LeMon5)

normal aberrant

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CD34-FISH Interim Study Results:

• Karyotype evolution detected in ~13% of pts. as yet

(median observation time of 9 months)

• Confirmation of evolution patterns known by CBA

• Delineation of new evolution patterns

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Combining FISH and SNP Analysis of CD34+ blood cells

Aims: • To increase number of informative cases

• To improve diagnostic accuracy

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Array

Affymetrix CytoScan HD Array

2.6 million markers – 750.000 SNPs – 1.800.000 CNV

100% of known cancer genes (526)

average marker spacing: 1,148 bp

250 ng DNA (own exp.: 100 ng possible)

3-4 days

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Pilot Study • N= 23 pts. • SNP-analysis of CD34+ blood cells (n=23) • Chromosome banding analysis of bm aspirate (n=22), pb (n=1) • FISH-analysis of CD34+ blood cells (n=21), bm (n=2) • Diagnoses: MDS: n=20, AML: n=3

• Results: # of abnormalities detected depending on the method

Conclusion:

•SNP + FISH on CD34+ pb feasible

•SNP-A increases the number of detectable abnormalities

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number of abnormalities

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Case Report 1

• Gain of genetic information

• Proof of clonality

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Case Report 1 • Suspected MDS RCMD, normal karyotype

TET2 Polymor-phism

• Proof of a microdeletion in 4q14 containing the TET2-gene (size 0.8 Mb) and a polymorphic deletion of 0.2 Mb proof of clonality

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Case Report 2


• Improvement of prognostication

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Case Report 2 (MDS RAEB-II)

• Karyotype: 47,XY,+8 [18]/46,XY [7]

• FISH-analysis of CD34+ blood cells: • +8 in 56 % of

interphase nuclei

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Case Report 2 (MDS-RAEB-II)

Consequence: • Worse prognosis with +8

and additional UPD 7q

UPD (7)(q11qter)

CD34+ bm cells

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Case Report 3


• Dissection of the molecular anatomy of a ring chromosome 11

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Case Report 3: Secondary AML after MDS Karyotype: 46,XX,del(5)(q13q33),r(11)hsr(11)(q23)

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• FISH-analysis of CD34+ blood cells: • 5q- in 98 % of interphase

nuclei

• MLL amplification in 93%

of interphase nuclei

Karyotype: 46,XX,del(5)(q13q33),r(11)hsr(11)(q23)

EGR (5q31) D5S23, D5S721 (5p15.2)

MLL telomeric MLL centromeric

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Combining FISH and SNP Analysis of CD34+ blood cells

diploid

amplification

deletion

Delineation of the molecular anatomy of a ring chromosome 11 with different regions of closely connected small deletions and amplifications of different degrees (3x - >10x) („chromotrypsis“) this case has complex abnormalities: prognosis: intermediate very poor

MLL

Dissection of ringchromosome 11

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Combined Sequential FISH + SNP-Study

“Sequential comprehensive genetic analysis of circulating CD 34+ cells in MDS” Start: Spring 2013

Observation period

24 months

MDS, low- and int-1 according to IPSS,

n= 75 pts, Enrollment period: 12 months

Cytogenetic analysis of peripheral blood cells

every three months

CD34+FISH-analyis

Cytogenetic analysis of bone marrow cells

according to guidelines

CD34+SNP-array-analysis

Chromosomal banding analysis

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Acknowledgements CD34 FISH- project: F. Braulke Cytogenetics/FISH/24 colour FISH: K. Shirneshan SNP-Array-Analysis/ CD34 FISH + SNP- project: C. Ganster Cytogenetic Prognosis: J. Schanz Lab technicians: R. Steffens, C. Schütze, C. Schulte, A. Kaufmann, S. Raimer-Probst, A.-K. Künzel, M. Neu, C. Wachenhausen, A. Tsetoulidis, A. Kominowski, B. M. Wendt, A. Gutermuth

International collaborators: F. Solé, M. Mallo, Barcelona G. Mufti, A. Mohamedali, London H. Tuechler, Vienna CD34 FISH Study members: M. Metz Göttingen J. Seraphin, S. Detken Northeim K. Götze, C. Müller-Thomas München U. Platzbecker Dresden T.H. Brümmendorf Aachen U. Germing, A. Kündgen Düsseldorf G. Bug, O. Ottmann Frankfurt A. Giagounidis Duisburg W.-K. Hofmann, F. Nolte Mannheim A. Böhme Frankfurt P. Schafhausen, J. Isernhagen Hamburg K. Jentsch-Ullrich Magdeburg B. Schmidt München M. Lübbert Freiburg R.F. Schlenk Ulm W. Blau Berlin

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Reserve slides

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Do we enrich enough cells for FISH + SNP?

26 MDS patients

3*105 cells -> 600 ng DNA

1*105 cells -> 200 ng DNA

Leucocyte count (pb) before enrichment Leuc

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Karyotype evolution by CB and CD34+ pb FISH-Analysis

FISH-Analysis of CD34+ pb: − 5q- − -7 − TEL-loss (12p-) − TP53-loss (17p-) − 20q- − RB1-loss (13q-)

EGR1 D5S721

D7S522 (7q31) CEP7

RB1

ISCN-Karyotype: 44,XX,del(5)(q13q33),-7,del(12)(p13p11.2),-17,- der(17;20)(q10;p10) [23]/ 44,idem,der(3;6)(p10;q10) [2]

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Karyotype evolution by SNP-Analysis (CD34+ pb)

Month 0:

Loss UPD

ISCN-Karyotype: 44,XX,del(5)(q13q33),-7,del(12)(p13p11.2),-17,der(17;20)(q10;p10) [23]/ 44,idem,der(3;6)(p10;q10) [2]

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Karyotype evolution by SNP-Analysis (CD34+ pb)

Month 0:

Loss UPD

ISCN-Karyotype: 44,XX,del(5)(q13q33),-7,del(12)(p13p11.2),-17,der(17;20)(q10;p10) [23]/ 44,idem,der(3;6)(p10;q10) [2]

Evolution (Month 4): additional loss regression of 13q-

Sponsored by:






30 January 2013


UTILIZATION OF A SNP MICROARRAY FOR THE STUDY

OF LEUKEMIA

STUART SCHWARTZ, PHD, FACMG

Wednesday, January 30, 2013 12:00 P.M.

I am an employee of Laboratory

Corporation of America Holdings®

DISCLOSURE

VALIDATION AND CLINICAL STUDIES

• CLL - >225 patients • MDS - >800 patients • Others - >200 patients

• CMML, AML, ALL, MM and Tumors

• Will focus on CLL and MDS today • What will array analysis add to the evaluation

of these patients [INTERNAL DATA – DECEMBER, 2012]

Platform currently utilized in laboratory

More than 2.69 million markers ◦ Across the entire genome (spacing ~384 bp in ISCA genes)

743,304 SNPs (single nucleotide polymorphisms) ◦ Polymorphic probes ◦ Assessing copy number changes ◦ Assessing genotypes

1,953,246 structural probes (copy number probes) ◦ Non-polymorphic ◦ Assessing copy number change

AFFYMETRIX® SNP CYTOSCANTM HD

MDS STUDIES

NORMAL CHROMOSOME – ARRAY FINDINGS

ABNROMAL CHROMOSOMES/FISH – ARRAY ADDITIONAL INFORMATION

CYTOGENETIC ABNORMALITIES

50%

NORMAL 35%

ARRAY ONLY ABNORMALITIES

15%

INITIAL MDS STUDY– %ABNORMALITIES

30% OF NORMAL CHROMOSOME FINDINGS – ARRAY ABNORMALITY

NORMAL CHROMOSOMES/FISH ARRAY – DETECTED RUNX1 DELETIONS

PATIENT FINDINGS - IMPLICATIONS

Normal cytogenetics Patients ~1-2.6 Mb deletion of 21q22.11-q22.12

– Includes RUNX1 Implications of RUNX1 deletion

– Predictor of poor overall survival – Patients have an inferior event free survival – Resistance to therapy and inferior outcome

Only detected by array analysis

CLINICAL STUDY - MDS

Utilization of microarray for the routine study of patients to evaluate MDS

>800 patients studied Results Overall, when chromosomes were normal

Array studies revealed an additional 24.3% of patients with abnormalities

When chromosomes were abnormal, Array provided additional information in 40% - of the time Prognostic information in 36% of the patients obtained


Results When both flow cytometry and hematopathology

evaluation could not definitively confirm MDS Array analysis - abnormalities in 27.4% of the patients

When either flow cytometry and hematopathology evaluation were consistent with MDS Array analysis revealed abnormalities in 80% of the patients

51.5% of the patients with abnormalities in this study had segmental UPD The majority (54.9%) were as individual changes 39.2% were present with copy number change


Results Clonal evolution was seen in 21.2% of the patients Chromothripsis in 6.1% of patients

The most common changes seen involved the TET2 gene (either as a deletion of

UPD4q – 19.2% of patients, RUNX1 gene involved ~7.0%

MDS – (20q-) PATIENT STUDY ADDITIONAL INFORMATION

68 year old Initial diagnosis – persistent pancytopenia of unclear

etiology Flow analysis – no abnormalities Cytogenetic/FISH findings

Cytogenetic – 46,XY,del(20)(q11.2q13.3)[5]/46,XY[15] FISH – 20q- (2.8% of cells)

20Q DELETION DETECTED BY THE ARRAY ~15% OF DNA

CHROMOSOME 14 UPD; DISTAL ~75%; PROXIMAL 15%; UPD 14 – INITIAL ABNORMALITY; 20Q- CLONAL EVOLUTION

ARRAY - SIGNIFICANCE

Array findings Deletion 20q confirmed Uniparental 14 detected

Uniparental disomy Associated with homozygous mutation UPD 14 – shown to be the initial abnormality (driver of

disease) Clonal evolution detected

Deletion 20q Expansion of UPD

CLL STUDIES

NORMAL CHROMOSOME – ARRAY FINDINGS

ABNROMAL CHROMOSOMES/FISH – ARRAY ADDITIONAL INFORMATION

CLL PATIENT STUDY – NORMAL FISH

Patient with CLL No abnormality detected from routine FISH analysis

Array – duplication of 2p – Marker of progression and indicative of poor

prognosis – Not detected by targeted analysis

Acquired uniparental disomy (UPD) 17 – Suggestive of homozygous p53 mutation – Detectable by a SNP array

CLL – BETTER DELINEATION BY ARRAY ANALYSIS

64 year old male Flow: antigenic profile characteristic of chronic

lymphocytic leukemia (CLL) ~30% of total CD38 expression – not detected ZAP70 expression – not detected FISH: Only 13q deletion (72.5%) These are all better prognostic factors

13q- INCLUDING RB1 AND MIR15/16

SEGMENTAL UPD FOR 17P DETECTED

ARRAY ANALYSIS

Array analysis demonstrated three abnormalities 13q deletion (includes Rb1 and mir15/16) - ~55% 11p deletion - ~55% aUPD 17p - ~80%

Segmental UPD of 17p – predominant finding Findings suggest association with an

adverse prognosis

CLL PATIENT – GENOME COMPLEXITY

89 year old male FISH Trisomy 12 13q deletion

Flow: 89% of leukocytes with a B-cell chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL) phenotype

Morphology – consistent with CLL/SLL

CHROMOSOME 13 – GENOME INSTABILITY NUMEROUS DELETIONS - CHROMOSOMTHRIPSIS

NO GAIN OR LOSS OF GENETIC MATERIAL UPD – 19Q

CLL PATIENT – GENOME COMPEXITY

FISH findings Intermediate

But array Complex lymphoid evolution Two clones

100% and 85%

Chromothripsis Uniparental disomy Genome complexity

24 of 25 patients had normal chromosome findings 21 of 24 had abnormalities by the array (87.5%)

7 (of 25) patients had both normal chromosomes and FISH 4 of 7 had abnormalities by the array (57.1%)

18 (of 25) patients had abnormalities by chromosomes/FISH All (100%) had additional abnormalities detected by

the array

MULTIPLE MYELOMA – RESULTS OVERVIEW

MULTIPLE MYELOMA – CCND1+

5 patients Normal karyotypes FISH – Extra signal CCND1+

Based on FISH – Trisomy 11/Hyperdiploid Better prognosis?

Based on array Two patients – Hyperdiploid; better prognosis Three patients – Unfavorable prognosis

MULTIPLE MYELOMA – CCND1+

Array reveals complex abnormalities Hyperdiploid Multiple gains and loss

1p- and 8q+ (MYC)

Chromothripsis Multiple gains and losses Chromosomes 1 and 12

Uniparental disomy Chromosomes 13 and 16

Overall Unfavorable prognosis

ACQUIRED UPD

Segmental UPD – important and prevalent in a variety of leukemias

Over 120 patients with UPD identified Seen overall in ~15% of patients

40% - CMML 45.5% - Tumors

Seen as isolated anomaly ~54% of the time Majority was segmental and could be detected with

as little as 10% DNA

ACQUIRED UPD - IMPLICATIONS

• Important and frequent finding in cancer • Only detectable with genotyping array • In some patients associated with poor course • Most common aUPD regions seen in MDS: 4q, 9p,

11q, 17p • Thought to provide mechanism to duplicate existing

mutation • In TET2, JAK2, CBL,TP53, respectively

Advantages Delineation of all abnormalities in the genome

Clear demarcation of breakpoints – genes involved

Detection of: Uniparental disomy Homozygous deletions Chromothripsis

Detection of abnormalities not available by FISH and not seen in chromosome analysis

Ability to better provide better prognostic information

BENEFITS OF ARRAY

Disadvantages Cannot pick up balanced translocations Mosaicism – FISH more sensitive than array

However – have been able to detect 7% mosaicism Close to sensitivity of FISH

ARRAY - DISADVANTAGES

DIAGNOSTIC/PROGNOSTIC

As discussed initially – array provides additional information – which can be used for better prognosis

Prognostic/diagnostic information obtained by SNP array in a variety of cancers CLL (Abnormal) - 32.2% MDS - 37.1%

OVERALL IMPLICATIONS

Microarray should be used in any leukemia (CLL, MDS, AML, ALL or solid tumor) when cytogenetics and FISH are normal – For both prognosis and diagnosis

Microarray can be used in abnormal patients with CLL and MDS to better define prognosis Also – AML, ALL, CML

Only SNP array can detected UPD

CONCLUSIONS

Detects both copy number and copy neutral changes Detection of abnormalities in cytogenetics/FISH normal

patients Dosage levels correlate with progression/clonal evolution Resolve complex karyotypes, cryptic rearrangements Unidentifiable chromosomes Wrong assumptions

Detection of additional abnormalities in >60% of patients Detection of complexity Provides diagnostic/prognostic information

ACKNOWLEDGEMENTS

LabCorp Peter Papenhausen Jim Tepperberg Inder Gadi Rachel Burnside Vikram Jaswaney Karen Phillips Hiba Risheg Liz Keitges Rao Potluri Romela Passion Brooke Rush Holly Taylor

LabCorp - Array Lab ◦ Mike Phelan ◦ Brian Williford ◦ Carolyn Bullen ◦ Jessica Whaley-Davis ◦ Renee Royster ◦ Laurelin Cottingham ◦ Colin Osborne ◦ Jeff Meitz ◦ Chris Younger ◦ Christina Goss ◦ Savanna Schepis

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30 January 2013


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30 January 2013


PLEASE STAND BY… the webinar Genetic Biomarkers Revealed...G-CCC. CD34-FISH •Circulating CD34+ cells show the same chromosomal aberrations as bone marrow cells (Haase et al., Blood,

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