Pergamon Lmkmia Research Vol. 21, No. 9, pp. W-809. 1997.

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SYNTHESIS OF QUANTITATION STANDARDS FOR NESTED COMPETITIVE PCR FOR THE DETERMINATION OF MINIMAL RESIDUAL DISEASE IN B-LINEAGE ACUTE LYMPHOBLASTIC

LEUKAEMIA

Gerald Martin* and Robert J. Mairst

*Department of Haematology, University of Cambridge, MRC Centre, Cambridge CB2 2HQ, UK and TDepartment of Radiation Oncology, University of Glasgow, Bearsden, Glasgow G61 IBD, UK

(Received 28 March 1997. Accepted 23 April 1997

Abstract-Quantitative PCR-based assays of leukaemic ceils in remission marrow hold promise for therapeutic guidance, but are not yet sufficiently reliable for clinical application. For B-lineage ALL, these assays usually involve PCR of clonal somatic gene rearrangements of the immunoglobulin heavy chain gene. The most accurate quantification can be achieved by competitive PCR. Here we present a novel approach for the production of reference standards for use in nested competitive PCR of these gene rearrangements, which might enable more reliable assessment of MRD for prognosis and selection of patients for individualised therapy. 0 1997 Elsevier Science Ltd

In recent years the emphasis of research directed against the detection of minimal residual disease in childhood and adult acute lymphoblastic leukemia (ALL) has shifted towards the use of highly sensitive cellular and molecular biology techniques, including in vitro cell culture, multiparameter flow cytometry, and the poly- merase chain reaction (PCR). In the case of B-lineage ALL, PCR usually involves the use of clone-specific primers for the amplification of rearranged antigen receptor genes (immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) genes) [l-4]. Recent PCR studies have suggested that not only the presence or absence of detectable leukemia, but also the level of detectable leukaemia may be useful prognostic indicators, allowing for individualization of treatment [2].

At present the most sensitive PCR-based methodol- ogies can detect one leukemic cell in one million normal bone marrow mononuclear cells [5]. However, quantita- tion lacks precision, impeding clinical application of the PCR. This is partly because the method relies rely on separate amplification of dilute diagnostic material to provide a standard curve against which signal from amplified DNA from remission samples is compared. The most reliable quantitation is achieved by competi- tive PCR (cPCR), which involves co-amplification of an internal reference standard with the same primer recognition sites as, and similar internal DNA sequences to, the target [6]. Because the target and reference sequence bind the same primer sequences, and are extended with equal efficiency, amplification of target

and reference is directly proportional to the initial relative amounts of each species in the reaction vessel. In addition, the competitor molecule provides a control element to the assay-the absence of both competitor and clone-specific target indicating the presence of PCR inhibitors (unpublished observation). Co-amplification reference sequences can be generated by various strategies, including PCR-mediated deletion-insertion [7], in vitro mutagenesis [S], cross-species amplification [9], amplification of intron-containing DNA [lo], and ligation [ 111.

We recently described a methodology for the genera- tion of reference standards for the quantitation of minimal residual disease in acute lymphoblastic leuke- mia [ 121. This involved sequencing the clone-specific, highly variable, complementarity determining region III (CDRIII) of the patient’s leukemic cells. A single- stranded PCR primer derived from this sequence information was rendered double-stranded by the activity of DNA polymerase, and then ligated it to the blunt-ended Jn-containing restriction fragment of the IgH rearrangement from the B-lineage leukemia cell line Daudi. After PCR amplification using Jn and the clone- specific primers, the quantitation reference was identi- fied by agarose gel electrophoresis, and isolated. A further round of DNA polymerase activity, ligation, amplification and isolation resulted in the production of a reference sequence containing a consensus VH region primer at one end and the JH consensus primer at the other, so that this new quantitation standard could be

807

808 G. Martin and R. J. Mairs

1. Digest V,3-J,, amplified Dudi IgH rearrangement with Hue III enzyme.

I I J, end

2. Save J, end for ligation to clone-specific primers for all subsequent ALL patients.

I J, end

3. Amplify and sequence patient IgH rearrangement. Design clone-specific primer and V&lone-specific combination primer.

- - - - - - -

V, sequence, spacer, clone-specific sequence

4. Render V&lone-specific combination primer double stranded by DNA polymerase.

m:::.r:_B

V, sequence, spacer, clone-specific sequence

5. Ligate double-stranded V&lone-specific combination primer to saved Dmdi fragment.

B:::::-w

V”

4

J”

6. Amplify above with V, and J, primers, and isolate and quantify product.

Fig. 1. Flow chart of steps involved in the generation of clone-specific standards for nested competitive PCR.

used in a nested cPCR system in conjunction with a second (internal) consensus JH primer (J&), thereby improving the sensitivity and specificity of the assay.

We have devised a streamlined approach using a combined VH region/clone-specific primer. In addition to the consensus Vn region and Jn/JnI* primers (used in the original determination of CDRIII sequence and the first and second rounds of amplification in nested cPCR) and the clone-specific primer (used in the second round of nested cPCR), another primer-which combines the Vn region and clone-specific primer-is designed. This is then used to produce a V&lone-specific nested reference standard in a single polymerase/ligation/ amplification/isolation cycle in conjunction with the consensus JH primer (Fig. 1). The V&lone-specific primer is rendered double-stranded by the activity of DNA polymerase. It is then ligated to the blunt-ended HueIII restriction fragment of the Daudi cell line IgH rearrangement, and amplified using Vn and Jn con- sensus primers. The amplified product is then isolated from agarose gel following electrophoresis, and quanti- fied by absorbance at 260 mn. Materials and methods for these steps, and the sequences of PCR primers, have been described previously [ 121.

The prospect of applying clone-specific PCR-based methods to the clinical laboratory as part of the

evaluation of patient remission status raises a number of issues-not least the cost of these tests and their clinical relevance. In addition to reagent, equipment, and staffing costs, a licensing fee for the use of the PCR technology must also be paid. Will health services be able to afford the expense of satisfying a continuous requirement for new primers? One possible way to reduce cost would be to establish centralized primer production facilities, to specialize in leukemia-specific primer production. Bulk synthesis of consensus primers would help to standardize techniques and results between centers while expertise gained in primer design and use could benefit the design of primers for newly diagnosed patients. Only further research can determine the potential value of PCR-based detection methods in the clinic.

In conclusion, we have described a novel approach for the development of quantitation standards for use in nested competitive PCR assays of minimal residual disease in B-lineage ALL. Every standard is patient- specific, but can be generated quickly without excessive expense. The use of a competitor molecule not only allows quantitation, but also provides an element of test self-diagnosis, indicating the presence of PCR inhibitors within the patient sample.

Synthesis of quantitation standards 809

Acknowledgements-This work was supported by the Scottish Hospital Endowment Research Trust and the Cancer Research Campaign.

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