BCR/ABL1 Quant (CE IVD): a

Cost Effective Multiplex Assay

for Quantitative/Qualitative

Analysis of BCR/ABL1

Jane Markley

Ninad Pendse

Agenda

• BCR/ABL1 Quant (CE IVD) Assay

– Technology and Features

• BCR/ABL1 Quant (CE IVD) Assay

– Workflow and Procedures

• ARQ IS Calibrator Panel

2010 ASH Leukemia Advisory Board Findings

• Leukemia Ad Board participants included 9 KOLs from

leading academic centers and top reference labs, (3 IRIS

trial sites)

• When asked to rank criteria for a BCR/ABL test there was

consensus on top 3

– Sensitivity of 1/100,000. Sensitivity needs to be reported on patient

report.

– Reliability of test particularly at the low levels

– Harmonization and reporting of results on IS. Agreed that IS

standardized assay was appealing to them for use in their clinical

practice.

• Also noted were cost efficiencies.

CE IVD

BCR/ABL1 Quant (CE IVD) Assay• 3 color Multiplexed TaqMan® assay for detection and quantification of:

• BCR/ABL1 fusion transcripts (b2a2, b3a2, and e1a2)

• ABL1 (an endogenous control)

• BCR/ABL1 Quant Norm (an exogenous control)

• e1a2, b2a2, b3a2 subtype identity can be determined by CE fractionation

• Internal and external assay calibration powered by Armored RNA Technology provides

comprehensive coverage and eliminates the need for reflex testing

• UNG compatible for amplicon contamination prevention (optional)

• Includes AmpliTaq Gold®, RT ENZYMES and freedom to operate (Roche)

• Includes data analysis software (ratio, absolute copy number)

• Manufactured in the US in compliance with ISO 13485 and cGMP Quality Systems

Assay Components

RT reagents (48 rxn)

– RT enzyme mix

– RT buffer mix

– Reaction diluent

PCR reagents (48 rxn)

– qPCR enzyme

– qPCR buffer mix

– qPCR primer/probe mix

– Reaction diluent

Controls

– Exogenous extractable process control: ARQ Norm

– Calibrators: 4 ARQ blends to build 4 point standard curves for BCR/ABL1, ABL1 and Norm

– Calibrator diluent

BCR/ABL Quant Analytical Sensitivity

1.Sensitivity

– Analytical performance studies with BCR/ABL1 Quant RUO show

excellent analytical sensitivity in cell line with detection of 1 leukemic

cell in 100,000 normal cells.

– A very sensitive assay will minimize the chance of false negative

results and improve robustness at very low BCR-ABL1 copy

number.

Preliminary Performance - Utilizing IVT Target RNAs†

• >6 Logs of linear dynamic range†

• LOQ=50 copies, LOD=10 copies

† Preliminary research data. The performance characteristics of this assay have not yet been established.

Brown J. T. et. al., Blood Cancer Journal, 2011

In this study, b2a2 positive cell line RNA was diluted into HL-60 RNA spiked with

1,000 copies of BCR/ABL1 Norm control

• Detects 1 BCR/ABL1 (+) Leukemia cell in 100,000 normal cells†

Analytical Sensitivity and Linear Dynamic Range†

† Preliminary research data. The performance characteristics of this assay have not yet been established.

>5 Log linear dynamic range†

LOQ=1:100,000 (0.001%)

BCR/ABL Quant and Armored RNA

2. Reliability

Armored RNA Technology allows for the utilization of

nuclease-resistant, RNA based calibrators and controls.

– The use of RNA-based calibrators provides a more accurate

calibration of an RNA-based target resulting in more accurate and

reliable quantitation of BCR-ABL1 and ABL transcripts.

– The use of a nuclease-resistant RNA based and extractable process

control provides controls for variability in the extraction, reverse

transcription and amplification steps of the test protocol resulting in

greater result accuracy and reliability.

• A patented and proven technology to manufacture and stabilize

RNA for use as:

• Positive/Negative control

• Process control (e.g., spike in non-target RNA)

• Assay calibrator (e.g., standard curves)

• External calibrator (e.g., proficiency panel)

• Nuclease resistant and stable in biological matrices

• Precisely quantified using a NIST-traceable reference standard

• Performance well established:

• In clinical molecular virology applications (HIV, HCV…)

• And now in clinical hematology applications

• Manufactured under cGMP to ensure lot-to-lot consistency

critical for reagents used in clinical molecular analysis

Armored RNAs Ideal Quantitative Standards

BCR/ABL1 Quant* - Internal and External Calibration

* Research Use Only, not for use in diagnostic procedures

Asuragen Advantage – Workflow efficiencies

3. Cost efficiencies

• Home brew and competitive assays use up a lot of the plate for running individual

calibrators, controls and samples

• Asuragen multiplex uses only 7-11 wells for controls and calibrators, leaving up to 89 wells

on 96 plate for sample testing.

• Kit includes all reagents under one lot number.

• CE identification of fusion transcripts makes the assay 2 in 1.

Plate Layout Illustrates Significant Cost Savings

Examples of Customer Feedback

• BCR/ABL1 Quant improved workflow, number of specimen tested per run and overall throughput.

• Inclusion of e1a2 resolved previously discrepant results between FISH and LDT (these specimens had to be sent out for further analysis).

• In regard to tech time and ease of use, there is much less manual

manipulation of tubes, samples and reagents in both the RT set up and real

time PCR plate set up. This includes fewer pipetting steps, most of which

utilize a multi-channel pipette.

• The work flow is very streamlined and is amenable to include many more

patient samples for only 10 wells of standard curve.

• The first actual assay I tested included approximately 40 patient samples. I

would never attempt to perform 40 RTs at one time with our current

procedure. We use a 96 well plate but can only test 9 patient samples at a

time.

• Studies show a close correlation in patient ratios between the two assays. This could lead to a seamless transition between the two assays.

• The assay includes the e1a2 breakpoint for which we are getting numerous requests (currently sent out).

ARQ IS Calibrator Panel:

Design and Characteristics

ARQ IS Calibrator Panel Utility

Ultimately, the ARQ BCR-ABL1 International Scale Calibrator Panel will

provide utility in the following applications:

• Routine monitoring of drift for assays already aligned to the IS through patient

specimen exchange with an IS reference laboratory to establish/validate an

IS conversion factor.

• Routine monitoring of assay performance, including verification of absolute

BCR-ABL1 and EC gene copy numbers, % ratio on the IS, and analytical

sensitivity and linearity.

• Reduce or eliminate the need for laboratories to perform patient sample

exchange with a National Reference Laboratory to align or maintain

alignment of Laboratory Developed Tests or Commercial Assays to the

International Scale.

ARQ IS Calibrator Panel Design

• Secondary reference material IS-standardized by direct comparison and

calibration to the WHO primary standard (White et al. Blood 2010)

• The ARQ IS Calibrator Panels will consist of blends of Armored RNA Quant

molecules (ARQ) covering a range of BCR-ABL1 to control gene % ratios

on the IS that represent the range of expression levels encountered in

clinical specimens, including CCyR (1%) and MMR (0.1%).

• Traceable to an analytical NIST phosphate standard to ensure formulation

precision, lot-to-lot consistency and maintenance of accurate BCR-ABL1 to

endogenous control gene ARs and known copy number values

% Ratio

Sample ABL1 BCR e13a2 or e14a2 ABL1 BCR

Calibrator 1 100,000 100,000 10,000 10 10

Calibrator 2 100,000 100,000 1,000 1 1

Calibrator 3 100,000 100,000 100 0.1 0.1

Calibrator 4 100,000 100,000 10 0.01 0.01

Neg Control 100,000 100,000 0 0 0

IS % Ratio

Sample ABL1 BCR

08/198 10.7 16.3

08/196 1.17 1.66

08/194 0.11 0.17

08/192 0.012 0.019

WHO Primary Standard Panel (e14a2) ARQ IS Calibrator Panels (e13a2 or e14a2)

ARQ IS Calibrator Panel Format

• Two Panels: 1 for e13a2 and 1 for e14a2

• Each Panel contains 4-level Calibrators and 1 negative control

Use of ARQ IS Calibrator Panels

Nominal IS % ratio

from WHO IS

reference panel

1. Test ~30 samples

at local lab

4. Repeat steps 1-3

to validate CF

5. Use CF until next

required validation(1 to 2 years or when local

method is changed)

1. Test 4-level

Panels at local lab

2. Compare nominal IS

% ratio and assess

linearity, slope and

analytical sensitivity

3. Continue to

routinely monitor

performance

Sample exchange method:

Time consuming and

infeasible for some labs

2. Test same

samples at IS

Reference Lab

3. Compare % ratio

to establish CF

Current Sample Exchange Method: Example

• Most % ratio measured by the local lab are above the equality line

indicating a bias in the results

• The bias and CF of the local lab relative to the reference laboratory

results is calculated as described in Branford et al. ( Blood 2008)

• In this example CF=1.6, i.e., % Ratio local Lab x 1.6 = IS % Ratio

• The CF will then be used by the local lab until next CF validation

-3

-2

-1

0

1

2

3

-3 -2 -1 0 1 2 3

Lo

g IS

% R

ati

o (

Re

fere

nc

e la

b)

Log % Ratio Local lab

Equality line X=Y

IS % Ratio generated

by the IS Reference

Laboratory

ARQ IS Calibrator Panel: Examples

Example 1 Example 2

• There is a small bias (local lab % Ratio is

about 2 fold higher than IS % Ratio)

• The local lab can correct % Ratio by dividing

the results by 2 (CF = 0.5) or choose to not

do so (e.g. lab already has a validated CF)

• Since the ARQ IS Calibrator Panel can be

included in subsequent runs, the local lab

can continue to monitor its bias

Equality line X=Y

• The % Ratio measured by the local lab are

as expected and close to the equality line

• There is almost no bias. The local lab is still

harmonized to the IS

• The ARQ IS Calibrator Panel can be

included in subsequent runs to monitor for

potential drift or run-specific issues

-3

-2

-1

0

1

2

-3 -2 -1 0 1 2

Lo

g IS

% R

ati

o A

RQ

Ca

lib

rato

r

Log % Ratio Local lab

-3

-2

-1

0

1

2

-3 -2 -1 0 1 2

Lo

g IS

% R

ati

o A

RQ

Ca

lib

rato

r

Log % Ratio Local lab

Equality line X=Y

y = 1.244x - 0.226

-3

-2

-1

0

1

2

-3 -2 -1 0 1 2

Lo

g IS

% R

ati

o A

RQ

Ca

lib

rato

r

Log % Ratio Local lab

Equality line X=Y

ARQ IS Calibrator Panel: Example 3

• There is a non-uniform bias (higher % Ratio at low BCR-ABL1 copy

number). This may be an assay-specific characteristic, or a run-specific

issue, or caused by a change in the method (e.g. new reagent lot)

• The local lab can flag this run or correct the % Ratio by using the linear

regression equation (e.g., patient at 0.12% is in fact in MMR at 0.04% IS)

• Again the local lab has the opportunity to continue to monitor its bias

0.12%

0.04%

-3

-2

-1

0

1

2

-3 -2 -1 0 1 2

Lo

g IS

% R

ati

o A

RQ

Ca

lib

rato

r

Log % Ratio Local lab

Equality line X=Y

ARQ IS Calibrator Panel: Example 4

• In this run, the low level ARQ IS Calibrator is not detected

• This method (or this run) is likely not sensitive enough to evaluate

residual disease below MMR (e.g. to evaluate CMR)

• This method (or this run) should not be used to report quantitative

results on the IS

Key features of ARQ IS Calibrator Panel

• Multiple IS % ratio levels for accurate comparison to the IS and an ability

to assess linearity and sensitivity.

• Include low positive sample to challenge analytical sensitivity of field

methods and protect the clinically relevant decision points for current

and next generation TKI (MMR and CMR)

• e13a2 or e14a2 panels available to challenge field methods that may not

have the same efficiency for both fusion transcripts

• Stable, nuclease resistant and made available to any local lab

• Can be tested with or without extraction

• Copy number of endogenous control gene representative of expression

levels in clinical specimens (unlike cell line-based material)

• Manufactured in cGMP environment, linked to NIST analytical reference

standard and tested with assay developed and manufactured in

compliance with QSR to ensure lot-to-lot consistency