Signature® LTx v2.

0 Kit:
Rapid Multiplex Detection
of Leukemia Fusion
Transcripts
Agenda

• Leukemia translocations

• Signature® technology platform

• Signature® LTx assay

• Representative examples:

- External evaluations

- Complementary role to cytogenetics

- Multicenter validation study

Leukemia and Molecular Classification
• Leukemia is a group of related malignancies of the blood cells
• Two broad categories: acute and chronic - many subtypes
• Characterization requires intensive laboratory studies:
- Morphology
- Cytochemistry
- Immunophenotyping
- Cytogenetics
- Molecular methods
• One of the most important features is the presence of specific balanced chromosomal
translocations
• In some cases, translocations define the disease and the type of treatment, for example:
- All-Trans-Retinoic Acid for t(15;17)-positive acute promyelocytic leukemia (APL)
- Tyrosine kinase inhibitor for t(9;22)-positive chronic myelogenous leukemia (CML)
Common Methods for Detection of Translocations
• Cytogenetics
- Globally determination of genetic abnormalities, both balanced and numerical
- Require dividing cells
- Can miss cryptic sites
- Not very sensitive (10-20%)
• FISH
- More sensitive (5-10%)
- Will not detect small abnormality (micro deletion/insertion, mutations)
- Difficult to multiplex
• Reverse transcription quantitative PCR (RT-qPCR)
- Very sensitive (<0.01%): minimal residual disease monitoring
- Rapid but multiplexing is very limited
- High cost if testing multiple targets
• Multiplex RT-PCR
- Size based detection: limited multiplexing and potential issue with non-specific products (gel, capillary electrophoresis)
- Hybridization base detection: limited throughput or high cost (microarray)
- Liquid bead array detection:
• Hybridization confirms sequence
• Multiplexing and throughput compatible with many applications
• Can be sensitive when optimized (0.1-1%)
Signature® Technology Platform

• Multiplex (2-80 analytes)

• 96-well plate format PCR or RT-PCR plate

• Streamlined workflow

• Rapid TAT (4-6 hours)

• Flexible platform Hybridization/detection plate

• Easy data interpretation

Results
(LIMS)

Digital Data Output

(MFI)
 Signature® Leukemia Workflow*

**

**No wash step required

*For Research Use Only.

Not For Use in Diagnostic Procedures.
 Signature® LTx v2.0*
• Multiplex RT-PCR for 9 common leukemia-associated translocations
(12 fusion transcripts)
• Simultaneous detection on a liquid bead array platform (Luminex)
• Incorporation of an endogenous GAPDH internal control
• 24 reactions, including the controls
• UNG compatible for amplicon contamination prevention (optional)

RT reagents Controls
– Enzymes – No template control (no RNA target)
– Buffer – Negative control (only GAPDH target)
– RT primer – Positive control (all RNA targets,
including GAPDH)
PCR reagents
– Buffer Hybridization
– PCR primer mix – Bead mix
– Buffer and conjugate

*For Research Use Only.

Not For Use in Diagnostic Procedures.
 Signature® LTx v2.0 Panel*
Classification Translocations Genes Involved/Fusion Transcripts
BCR-ABL1 e13a2
CML t(9;22)
BCR-ABL1 e14a2
t(9;22) BCR-ABL1 e1a2
t(1;19) TCF3-PBX1 (E2A/PBX1 e13e2)
ALL t(12;21) ETV6-RUNX1 (TEL/AML e5e2)
MLL-AFF1 (MLL/AF4 e9e5)
t(4;11)†
MLL-AFF1 (MLL/AF4 e10e4)
PML-RARA bcr1 (L form)
APL t(15;17)
PML-RARA bcr3 (S form)
CBFB-MYH11 A type
inv(16)
AML CBFB-MYH11 D type
t(8;21) RUNX1-RUNX1T1 (AML1/ETO e5e12)

†Signature LTx v2.0 Kit can detect two t(4;11) fusion transcripts, e10/e4 and
*For Research Use Only. e9/e5, but does not discriminate between them
Not For Use in Diagnostic Procedures.
 Analytical Specificity*

e1a2 b2a2 b3a2 AML/ETO Inv16-A Inv16-D RARa-L RARa-S TEL/AML E2A/PBX MLL/AF4 GAPDH
Neg Control 74 155 183 30 80 72 104 98 64 120 147 3718
Pos Control 1705 3924 2506 3708 3031 2237 4761 3425 2169 1779 3549 3011
NTC 98 139 54 82 48 84 77 119 35 38 95 55
e1a2 2994 109 51 66 151 103 94 154 97 75 40 3535
b2a2 84 5593 9 51 118 82 80 103 113 56 105 3445
b3a2 43 61 4723 150 61 48 77 145 98 103 146 2744
AML/ETO 41 0 22 6290 43 45 98 30 53 230 137 3306
inv16A 49 72 59 126 3790 29 46 68 95 84 172 2858
inv16D 64 76 79 51 66 3724 118 89 58 68 178 4315
RARa-L 56 91 85 140 88 41 5841 177 38 126 48 4080
RARa-S 78 84 21 7 84 154 82 3998 50 71 73 4016
TEL/AML 75 68 75 88 27 98 7 90 4047 93 38 4129
E2A/PBX 68 44 0 25 96 92 82 122 103 4621 168 3099
MLL/AF4 e9/e5 55 84 29 49 113 88 150 52 76 64 3832 3465
MLL/AF4 e10/e4 134 98 123 74 105 146 87 135 124 80 2668 4089

“One well, twelve results”

12 different fusion transcripts detected (400 ng total RNA input)
*For Research Use Only.
Not For Use in Diagnostic Procedures.
 Analytical Sensitivity (in cell lines)*

b2a2 b3a2 e1a2 E2A/PBXPML/RaRa-LTEL/AML AML/ETO MLL/AF4 Inv16-A GAPDH

HL60 134 138 135 175 144 144 45 158 114 4780
NTC 115 78 153 94 128 113 81 190 80 106
BV173 3632 114 140 81 104 65 60 108 77 4425
10% (100 ng in 900 ng

K-562 149 3976 184 106 106 77 90 169 85 4274

SupB15 89 116 2340 92 112 112 86 162 95 4442
697 108 130 148 2821 84 113 103 126 64 4421
HL60)

NB4 103 98 137 124 2239 87 65 75 151 4600

REH 89 166 73 124 78 2605 118 145 113 4494
Kasumi1 98 89 146 90 125 96 5805 197 40 4569
RS4 133 131 182 105 97 71 71 3241 76 4829
ME-1 72 96 107 113 123 58 59 126 4999 4330
BV173 2019 99 51 107 90 74 108 92 41 4532
1% (10 ng in 990 ng

K-562 122 2045 80 83 104 100 60 78 59 4410

SupB15 43 93 553 35 51 6 151 114 61 4341
697 93 130 110 1144 80 48 45 148 44 4478
HL60)

NB4 135 121 87 155 702 100 75 165 28 4630

REH 80 102 40 89 84 952 96 178 67 4550
Kasumi1 85 53 149 87 65 87 3728 134 76 4490
RS4 11 114 33 94 0 87 78 811 63 4625
ME-1 94 72 109 127 84 82 87 144 2129 4628

• Sensitivity reached at least 1% using cell line dilutions

• Equivalent to about 1 positive cell in 100 negative cells
*For Research Use Only.
Not For Use in Diagnostic Procedures.
Key Assay Attributes
• Multiplex RT-PCR for 11 common leukemia associated fusion
transcripts
- Leads to faster turn-around time than conventional methods

• Small speimen input volume (from 1 mL of whole blood or

100uL bone marrow)
- Reduces minimum collection volume requirements

• Simultaneous detection on a liquid bead array platform

- Reduces consumables and reagent cost
External Evaluation at MD Anderson
Conclusions from MD Anderson Research Study

• Conclusions
- “The Asuragen LTx complete panel multiplex bead-array assay correctly
identified fusion transcripts present in diagnostic specimens
- Useful for rapid screening and classification of newly diagnosed leukemias
but not for specimens with less than 15% tumor cells
- The assay has distinct advantage over available single analyte assays
when limited diagnostic specimen is available (~500 ng of total RNA is
sufficient)
- Eliminates separate assay set up for internal control
- Cost effective
- Easy to interpret the data”
Complementary Role to Conventional Cytogenetics (CC)

Poster presented at AMP 2010

HUP Research Study Conclusions
• This Study found a false negative rate of 12% for CC for the translocations included
in MRP

• In cases with an adequate RNA sample, MRP was able to detect translocations in all
50 cases detected by CC

• Signature LTx is useful when CC is not technically feasible

• Use of both CC and MRP assay in a complimentary manner is essential for

comprehensive evaluation of acute leukemias
Publication: King et al., Am J Clin Pathol 2011
 Evaluation of CE-Marked IVD Signature LTX*

Poster presented at
Planet xMAP 2011

*Available only in Europe

Multicenter Validation Study

Poster presented at AMP 2011

Manuscript in preparation