Reference Materials and BCR-ABL Quantitation

Laboratory Technology

Standardization and high-level analytical performance are two key objectives of molecular pathology laboratories to achieve consistent interpretation of individual patient response across time and geography.

The need for standardization is even greater for markers used routinely to monitor molecular response to well-established treatment options, such as BCR-ABL for chronic myelogenous leukemia (CML) patients treated with tyrosine kinase inhibitors.^1,2 Various RQ-PCR protocols have been developed to quantify BCR-ABL based on single or double plasmids, different primers, probes or control genes (ABL, GUS, BCR). However, substantial variation in BCR-ABL quantitation has been observed across laboratories, with up to 5-fold changes compared to reference value.^3,4

International Scale and Reference Materials

Over the last decade, following the progressive introduction of several tyrosine kinase inhibitors, CML experts have been working to improve standardization and accuracy of BCR-ABL quantitation. An International Scale (IS) was defined and established an absolute value of 0.1% BCR-ABL for Major Molecular Response (MMR).⁵ Obtaining MMR in a particular time frame is now considered to be a major treatment objective, which has potential consequences for treatment strategies.

Click to view Figure 1.

Following the definition of the IS, a system made up of reference laboratories and quality assurance/proficiency testing networks was put in place to provide molecular labs with an individual conversion factor, allowing labs to convert their own results to the IS.³

However, this system, based on regular exchanges and quantification of sets of CML patient samples between a reference site and the molecular lab, is not widely available in all countries and requires complex, time-consuming and expensive procedures.⁶

To address these limitations, reference materials have been recently validated under the World Health Organization (WHO) umbrella. The WHO reference panel is comprised of four cell dilutions corresponding to 10%, 1%, 0.1% (MMR value) and 0.01% BCR-ABL on the IS.⁵ These calibrated reference materials, produced and maintained in limited quantity-3,500 vials per dilution-are intended to be used by assay manufacturers to develop secondary reference materials that can be produced with no volume limitation.

Matching Highest Standards of Quality, Performance

In line with these recent developments and recommendations, Ipsogen has designed a new kit to quantify BCR-ABL p210 Mbcr: the BCR-ABL IS-MMR kit. This kit includes three new features:

• an RNA sample calibrated on the IS Primary Reference Materials (IS-MMR CAL), with an assigned batch-specific IS value close to 0.1% (see Fig. 1 for IS-MMR manufacturing process),

• a single plasmid containing both BCR-ABL and ABL as a control gene and

• four ABL standard dilutions and five BCR-ABL standard dilutions.

Performances of the new kit have been assessed in several analytical studies. Limit of detection is 0.0069% BCR-ABL, with a high analytical precision particularly around the MMR level (CV=25%). Linearity of the assay has been demonstrated between 0.003 and 65% BCR-ABL, allowing the use of a single calibrator to convert assay results to the IS.

The objective of incorporating the IS-MMR calibrator in each analytical run was to allow for an easy and accurate conversion to the IS. This has been demonstrated in an international multicenter study using the kit with the recommended reverse transcription and amplification enzymes (Ipsogen RT kit and ExTaq^®, Takara for RQ-PCR). Thirteen European centers and one U.S. center were involved in this trial testing of eight RNA samples with either the kit or their home-brew method.

Click to view Figure 2.

Despite the use of individual laboratory conversion factors (when available for routine practice), inter-laboratory variability was still significant with the home brew tests (only 70% of the measurements were in the 2-fold range), while inter-laboratory variability with the IS-MMR kit was in the 2-fold range for more than 90% of the measurements (Fig. 2). The kit features make it easy to use within the framework of routine lab activities, as illustrated by a low rate of technical failures. To further document the added value of the BCR-ABL IS-MMR kit, a multicenter North American study will soon be launched.

* In North America, Ipsogen products are not intended for diagnostic use and are for Research Use Only (RUO). In Europe and the rest of the world, Ipsogen provides CE marked and RUO products.

Dr. Peyro-Saint-Paul is chief medical officer, Ipsogen SA, Marseille, France.

References

1. NCCN CML Guidelines Updated 2011. Available at: http://www.nccn.org/network/business_insights/flash_updates/flash_update_information.asp?FlashID=8 (last accessed Feb. 24, 2011).

2. Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia: An update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009;27:6041-6051.

3. Branford S, Fletcher L, Cross NCP, Mller MC, Hochhaus A, et al. 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. Blood 2008;112;8;3330-8.

4. Zhang T, Grenier S, Nwachukwu B, Wei C, Lipton JH, et al. Inter-laboratory comparison of chronic myeloid leukemia minimal residual disease monitoring: Summary and recommendations. J Mol Diagn 2007;9(4):421-30.

5. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: Review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108;1;28-37.

6. White HE, et al. Establishment of the 1st World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood 2010 Aug 18.