LeumetaTM: (Plasma-based testing):
New Paradigm for Clinical Testing
in Leukemia & Lymphoma
Maher Albitar, MD
Quest Diagnostics, Nichols Institute
San Juan Capistrano, CA 92690
(949)728-4784
Maher.x.Albitar@Questdiagnostics.com
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Cancer is Not a Physiologic Process
•High uncontrolled proliferation rate
•High relative turnover of cells
•Heterogeneity within the tumor cells
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Circulating Cellular Debris Contains Fingerprints of Tumor Cells
Plasma
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Hypothesis
•Plasma (or serum) contains the “fingerprints”
of cancer cells
•Plasma (or serum) is enriched by tumorspecific DNA, RNA and cellular proteins.
•Every cell-based test can also be performed
using plasma (LeumetaTM).
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Why Hematologic Diseases?
•Neoplastic cells “swim” in plasma/serum.
•Hematologic diseases can be patchy
•Reticuloendothlial system is frequently
damaged in hematologic diseases
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Example of Heterogeneity in Bone Marrow in a
Patients with CML
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Marrow Fibrosis and Poor Aspiration
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Plasma-Based (LeumetaTM ) DNA Tests
•Mutations
•Chromosomal translocations
•Chromosomal aberrations (deletions, amplification)
•Minimal Residual disease
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Plasma is More Sensitive in Detecting JAK2 Mutation
and Homozygous/Hemizygous Patients
Patient #1
Forward
Cells
Reverse
Forward
Plasma
Reverse
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Patient #2
Patients with Homozygous/Hemizygous JAK2 as Detected
Using Plasma Have More Aggressive Disease
Blas ts <20%
Cumulative Proportion Surviving
1.0
0.9
0.8
0.7
P=0.05
0.6
0.5
P=0.5
0.4
He t JAK2 M utant (n=23)
0.3
He m i/hom ozygous JAK2 M utant (n=19)
0.2
Unm utate d (n=42)
0.1
0.0
0
26
52
78
104 130 156 182 208 234 260 286 312 338
Wee k s
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Plasma is More Sensitive than Cells in
Detecting TP53 Mutations
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Plasma is More Sensitive in Detecting Ras Oncogene
Mutations
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Detection of 5q Deletion in the Plasma of a
Patients with AML Using LOH
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Plasma is more Sensitive than Peripheral Blood Cells in Detecting
NPM1 Mutations in AML
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Plasma-Based (LeumetaTM ) RNA Tests
•Mutations
•Chromosomal translocations
•Expression and quantification
•Minimal Residual disease
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Plasma is More Sensitive than Cells in Detecting ABL
Kinase Mutations
931T>C (F311L)
758A>T (Y253F)
F
PB
R
F
BM
R
F
Plasma
R
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944C>T (T315I)
Plasma is More Sensitive in Detecting T315I Mutation
Using ASO Assay
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ID
TYPE
SEQ.RESULT % Mut of total
1003
PB
Wt
0
1003P
1004
PLASMA
BM
Wt
Wt
15
3
992P
PLASMA
Mut (T)
100
992
991
CELL
BM
C/T
C/T
53
61
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Plasma is More Sensitive in Monitoring BCR-ABL
Transcript in Patients with CML
BCR-ABL: ABL Ratio (Log)
10
1
0.1
0.01
0.001
1e -4
Ce lls
Plasma
1e -5
Bas e line
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3 M onths 6 M onths 9 M onths 12 M onths
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Plasma-Based (LeumetaTM ) Protein Tests
•Protein levels
•Phosphorylation and modification
•Enzymatic activity
•Tumor markers
•Diagnosis and classification
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Membrane Attachment of CD20 and CD52
CD52
GPI Anchor
CD20
N
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Carbohydrate
C
20
High levels of cCD20 in NHL but not in Hodgkin’s
Disease
Pre-therapy
1100
±1.96*Std. Err.
±1.00*Std. Err.
Mean
900
cCD20 (nM/L)
P<0.0001
700
500
300
100
HD (37)
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NHL (65)
21
Normal (15)
High Levels of cCD52 in NHL but not in Hodgkin’s
Disease
Pre-therapy
360
±1.96*Std. Err.
±1.00*Std. Err.
Mean
300
P<0.0001
cCD52 (nM/L)
240
180
120
60
0
HD (37)
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NHL (65)
22
Normal (15)
0.8
0.6
0.4
0.2
Probability of Survival
1.0
Shorter Survival in Patients with Lymphoma and High Levels
of cCD20
0.0
sCD20 M/ml
Pat.
<= 424.8
26
> 424.8
39
p-value = 0.008
0
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50
100
23
Died
14
32
150
Time (months)
200
0.6
0.4
Low b2m, Low CD52
Low b2m, High CD52
High b2m, Low CD52
High b2m, High CD52
0.2
Probability of Survival
0.8
1.0
Levels of cCD52 (23n Cut-off) and b2M (3.5 cut-off)
Are Useful in Stratifying Patients with CLL
0.0
p-value=<0.0001
0
1
2
3
Time (years)
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4
High Levels of cCD20 in CLL Correlate with Short
Survival
1.0
Cumulative Proportion Surviving
0.9
0.8
0.7
0.6
0.5
cCD20 (nM/L)
<1875
0.4
0.3
Pts
149
Died
30
>1875
19
P=0.01
7
0.2
0.1
0.0
0
10
20
30
Months
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40
50
60
70
Cell-Free Circulating Stem Cell Marker (CD34)
in Patients with Leukemia
cCD34 in AM L and M DS
Cumulative Proportion Surviving
1.0
0.9
cCD34(U/ul) Pats. Died
<10845 71
24
0.8
>10845
68
P=0.01
0.7
35
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
16
32
48
64
80
96
112 128 144 160 176 192 208
We e k s
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Cell-Free Circulating CD4 as a Biomarker in Patients
with Myelodysplastic Syndrome
cCD4 in M DS
Cumulative Proportion Surviving
1.0
0.9
cCD4 (U/ul) Pats. Died
<1029
19
13
0.8
>1029
25
P=0.04
0.7
11
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
16
32
48
64
80
96
112 128 144 160 176 192 208
We e k s
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Plasma Proteasome Activity as a Biomarker in Patients
with Acute Myeloid Leukemia
1.2
p = 0.002
1
Censored
Casp >= 2.4
Probability
0.8
Casp < 2.4
0.6
0.4
E/N=65/94
0.2
E/N=80/94
0
0
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100
200
Tim e of follow -up (w eeks)
28
300
400
Plasma BCR-ABL Protein in Patients with CML
(Pre-Rx)
BCR-ABL levels in Previously Untreated CML Patients
A
2.6e6
2.4e6
2.2e6
Mol/10 ul Plasma (100 beads)
2e6
1.8e6
1.6e6
1.4e6
1.2e6
1e6
8e5
6e5
4e5
2e5
0
0
2
1
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3
6
5
8
7
9
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53
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Plasma Levels of Phospho-BCR-ABL Protein in
Patients with CML (Pre-Rx)
B
Thr(735) and Tyr(245) phosphorylation in previously untreated CML Patients
2.4
Phospho:Total BCR-ABL Ratio
2.0
Phospho-Thr(735):BCR-ABL Ratio
Phospho-Tyr(245):BCR-ABL Ratio
1.6
1.2
0.8
0.4
0.0
0
2
1
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3
6
5
8
7
9
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53
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Currently Available Leumeta Tests
Plasma Cells
16031X/ 16029X abl Kinase Domain Mutation in CML
17679X/ 14991X t(11;14) bcl-1/JH(MCL) Real Time PCR
17690X/ 15007X t(14;18) bcl-2/JH Real Time PCR
17702X/ 15480X IgVH Mutation Analysis
16101X/ 16102X JAK2 Mutation (V617F)
16104X/ 16106X c-kit Mutation Analysis
16119X/ 14868X B-cell gene rearrangement, Qual., PCR
16118X/ 16005X B-cell gene rearrangement, Quant., PCR
16127X/ 16128X ras Mutation Analysis, Plasma-based,
17862X/ 15930X T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR
17861X/ 16025X T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR
17853X/ 15052X bcr/abl Gene Rearrangement, Quantitative PCR, Plasma-based
19783X/ 19782X ABL T3151 Mutation in CML
16159X/ 16158X NPM(Exon 12) Mutation Analysis
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Leumeta™ - Summary
New Paradigm in Testing
• Plasma contains the fingerprint of tumor cell
(Leukemia/lymphoma)
• Plasma is frequently more sensitive than cell
samples (less dilution by normal cells).
• Plasma-based testing reduce the need for biopsies
• Most of Plasma-based assays are quantitative,
easier to standardize and more objective.
• There is a need for the development of more
plasma-based tests to more significantly reduce
the need for biopsies.
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References
1.Manshouri T, Do KA, Wang X et al. Circulating CD20 is detectable in the plasma of patients with
chronic lymphocytic leukemia and is of prognostic significance. Blood. 101(7), 2507-2513 (2003).
2.Giles FJ, Vose JM, Do KA et al. Circulating CD20 and CD52 in patients with non-Hodgkin's
lymphoma or Hodgkin's disease. Br J Haematol. 123(5), 850-857 (2003).
3.Albitar M, Do KA, Johnson MM et al. Free circulating soluble CD52 as a tumor marker in
chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies. Cancer.
101(5), 999-1008 (2004).
4.Jilani I, Kantarjian H, Faraji H et al. Measurement of free circulating Bcr-Abl fusion protein and
its phosphorylation in patients with chronic myeloid leukemia. Blood. abstract 2006.
5.Rogers A, Joe Y, Dey A et al. Relative increase in leukemia-specific DNA in peripheral blood
plasma from patients with acute myeloid leukemia and myelodysplasia. Blood. 103(7), 2799-2801
(2004).
6.Ahmed M, Giles F, Joe Y et al. Use of plasma DNA in detection of loss of heterozygosity in
patients with multiple myeloma. Eur J Haematol. 71(3), 174-178 (2003).
7.Ma W, Kantarjian H, Verstovsek S et al. Hemizygous/homozygous and heterozygous JAK2
mutation detected in plasma of patients with myeloproliferative diseases: correlation with clinical
behaviour. Br J Haematol. 134(3), 341-343 (2006).
8.Ma W, Tseng R, Gorre M et al. Plasma RNA as an alternative to cells for monitoring molecular
response in patients with chronic myeloid leukemia. Haematologica. 92(2), 170-175 (2007).
9.Ma W, Kantarjian H, Jilani I et al. Heterogeneity in detecting Abl kinase mutations and better
sensitivity using circulating plasma RNA. Leukemia. 20(11), 1989-1991 (2006).
10.Ma W, Jilani I, Gorre M et al. Plasma as a source of mRNA for determining IgVH mutation
status in patients with chronic lymphocytic leukaemia. Br J Haematol. 133(6), 690-692 (2006).
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