Converting BCR-ABL1 to the International Scale:

Standardizing the Measurement of Relative Gene

Expression

Charles E. HillEmory University School of

Medicine

Outline

Measuring BCR-ABL1

The International Scale

Approaches for calibrating to the IS

Challenges of relative gene expression testing

Disclosures

• No financial interests/compensation from any of the companies discussed

• Assay kits were provided for evaluation by both Asuragen and Ipsogen

Chronic Myelogenous Leukemia

1.6 cases per 100,000

1.75:1 male:female

BCR-ABL1 translocation (Ph chromosome)

Molecular monitoring of BCR-ABL1 has become standard of care

SEER data 2012

Why test?

• BCR-ABL1 targeted by small molecules

• Prognosis

• Monitoring response

• Prediction of relapse

Monitoring BCR-ABL1Karyotype

Fluorescence in-situ Hybridization

PCR

RT-PCR

Competitive RT-PCR

Nested RT-PCR

qRT-PCR

Sensitivities

Karyotype – 1:20

FISH – 1-2:200

RT-PCR – 1:100,000

Response to Therapy

Hughes, NEJM, 2003, 349:1423

Prediction of Relapse

Press, Clin Cancer Res, 2007, 13:6136

NCCN Guidelines

• From NCCN 2.2013, qPCR should be performed:

• At diagnosis

• Every 3 months when responding to therapy. After CCyR, every 3 months for 3 years, then every 3-6 months

• For rising transcript levels (1 log) with MMR, repeat in 1-3 months

The IRIS Trial

• The International Randomized Study of Interferon versus STI571

• Major Molecular Response = 3 log decrease from average level at diagnosis

• Three laboratories harmonized results (Australia, UK, and USA)

BCR-ABL1 in Practice

• Many labs test for BCR-ABL1

• Testing and reporting are variable

• Many report change from diagnostic (local) baseline, but not calibrated the same

• Results not generally comparable between labs

Why is testing variable?

• Sample volume and integrity

• Processing and extraction

• Reverse transcription

• Control gene (BCR, ABL, GUSB, G6PDH, β2M)

• Primers

• Quantification standards

• Instrument and analysis

Harmonizing Results

• Median measurement of 30 shared baseline samples established as 100%

• 3 log decrease from baseline (0.1%) is Major Molecular Response

• For example, if median ratio BCR-ABL/BCR is 0.75, MMR = 0.00075

• International Scale defined by these two points

Getting to the IS

• 3 IRIS trial labs use IS by definition (Adelaide, Seattle, London)

• IRIS trial samples used to define 100% IS are not available to individual labs

• Sample exchange with a laboratory calibrated to the IS?

Pre-WHO Standard

• Branford, et al., Blood, 112:3330, 2008, “Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials,”

• Exchange of patient samples with IS calibrated reference lab to determine Conversion Factor

• Requires many samples and periodic re-assessment

• Very burdensome for few reference labs

Calibration Pre-WHO Standard

Branford, Blood, 2008, 112:3330

What does an IS do?

• An International Scale helps standardize quantification

• An IS does not improve pre-analytic issues

• An IS does not reduce inherent assay variability

• An IS does improve reporting

BCR-ABL1 WHO Standard

• 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR

• Intended for manufacturers/labs to develop secondary standards

• 4 ampules of freeze dried cells

• K562 cells (e14a2) diluted to different ratios in HL60 cells

• Spanning relevant range of %IS values

WHO BCR-ABL1 Standard

Secondary Standards

• Asuragen – ARQ IS Calibrators and BCR\ABL1 Quant

• Ipsogen – BCR-ABL Mbcr-IS assay

Asuragen ARQ IS Calibrator Panel

http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/ARQ_IS_CAL/arq_is_cal.aspx

Asuragen Calibrators

http://www.asuragen.com/Diagnostics/US/Products/qRT-PCR_Oncology/BCR_ABL1/

Ipsogen Calibrator

• Single point calibrator

• Tied to WHO Standard

• Plasmids

• Set to approximate 0.1% IS (MMR cutoff)

• Specific for Ipsogen assay

Correlation of two IS based assays

Patient Correlation

y = 1.0582x + 2.5081

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100Ipsogen IS %

Asu

rag

en I

S %

Comparison to Predicate Test

-1.6

-1.1

-0.6

-0.1

0.4

0.9

1.4

-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

Mean Log %

Dif

fere

nce

(IS

- p

red

icat

e)

Comparison to Predicate

• Very similar results for both calibrated to the IS

• IS tests are 0.4 and 0.35 lower than LDT

• Very important to communicate this to clinicians when changing reports

• Alternatively, report old result and IS

Advantages of Secondary Standards

• Allow calibration to WHO standard

• Simpler than exchanging many samples with reference lab

• Manufacturer’s QC and lot-to-lot control

• Periodic checks for drift are more feasible

Control Genes and Quantification

• ABL1 is the most commonly used control gene

• BCR was used as control gene for IRIS trial labs

• GUSB used by some

• EAC found ABL1, GUSB, and B2M suitable (Beillard, Leukemia, 2003, 17:2474)

• BCR was not reported in study by EAC

ABL1 as Control

Me

asu

red

% I

S

Log BCR-ABL1 Positive Cells

BCR as Control

Me

asu

red

% I

S

Log BCR-ABL1 Positive Cells

Other genes as Controls

• Do not participate in the translocation

• Should be expressed constitutively and not vastly over/under expressed compared to transgene

• GUSB and B2M

Limit of Detection

• Depends on transgene copies and control gene level

• What is minimum control gene for acceptable sensitivity?

• Calculate minimum control gene level to detect MMR?

“Complete” Molecular Response

Press et al, Clin Cancer Res 13, 6136 (2007)

CMR = no detectableBCR-ABL transcripts

Practical LOD Issues

• Balance sensitivity with not rejecting too many specimens

• For LOD=5 copies BCR-ABL, need 50,000 copies of control gene to get 4.0 log

• For 5.0 log need 500,000 copies control gene

• LOD is affected by total RNA input (5 copies in isolation easier to detect than 5 copies with background nucleic acid)

Summary

• The WHO Standard and development of IS have better standardized testing and reporting of BCR-ABL1

• Development of assays/calibrators tied to the IS make transition much simpler

• Are more sensitive assays needed?

Thank You

• Ruan Ramjit, Kaiser Permanente

• Karen Mann, Emory

• International BCR-ABL Standardization Group

• Emmanuel Labourier, Asuragen

• Mary Christopher, Ipsogen