Quantitation of the neoplastic cells using PCR

Until fairly recently, PCR amplification simply confirmed the presence (+) or absence (-) of tumor DNA sequences with little scope for quantifying the tumor bulk, particularly when using DNA as the PCR template. A band on agarose gel may represent the DNA from one cell—or many millions of cells. Clearly, this is of clinical importance if the information ob tained is to be of value in determining the need for further chemotherapy, which is the main rationale for attempting to detect MRD in the first place.

In the early years of PCR detection of MRD the starting template was usually DNA, but more recently PCR amplification of reverse-transcribed mRNA (termed 'complementary DNA' or 'cDNA') has been used. This refinement in PCR amplification has evolved where analysis of translocations such as t(9;22) or t(15;17) is impossible using a DNA template, simply because of the enormous size of the target being amplified. In these translocations the primer binding sites are so far apart on the DNA template that amplification is virtually impossible. However, the mRNA transcribed from these translocations undergoes considerable modification, with excision of introns making the mRNA counterpart of the translocation much smaller than the DNA.

Quantitation using competitive PCR templates has been possible for RNA-based PCR, and so we are able to quanti-tate the tumor cell burden in those diseases where RNA is the nucleic acid used for the PCR assays. Diseases in which reverse transcriptase PCR (RT-PCR) is possible, with quantitation of the tumor burden, include CML [with t(9;22)], AMLM3 [t(15;17)] and AML M2 [t(8;21)].

DNA templates are more difficult to quantitate, although competitive PCR templates may be of value here also. Recent technologies such as the TaqMan® real-time PCR machines may allow true quantitation using DNA as starting material. This system uses an internal oligonucleotide probe with added reporter and quenching activities (Figure 6.7). After primer and probe annealing, the reporter dye is cleaved off by the 5'-3' nuclease activity of Taq DNA polymerase during primer extension (Figure 6.8). This cleavage of the probe separates the reporter from quencher dye, greatly increasing the reporter dye signal. The sequence detector is able to detect the fluorescent signal during thermal cycling. The advantages of this system are the elimination of post-PCR processing and the ability to examine the entire PCR process—not simply the endpoint of amplification. Moreover, since the probe is designed to be sequence-specific, non-specific amplification products are not detected.

Primers

VH family

V

N

D

N

JH 1st amp.

ASO 2nd amp.

JH 1st amp.

Fig. 6.6 Semi-nested PCR of IgH region in patient with B-cell tumor

V- and J-region primers are used to generate the initial PCR product. Using DNA sequence information, an allele-specific oligonucleotide (ASO) primer unique to that patient is constructed and used with the V region primer to amplify an aliquot of the first-round PCR product.

Fig. 6.7 Real-time PCR amplification

See text for details.

Taq Pol

Taq Pol

Fig. 6.8 Standard curves generated for accurate quantitation of leukemic cell burden

Quantitation by real-time PCR requires generation of a standard curve. A known amount of template DNA is diluted into genomic DNA and amplified by PCR. The threshold cycle is the cycle number at which reported fluorescence is first detected above background and is proportional to the amount of starting template DNA. The threshold cycle number is then plotted against the known amounts and a standard curve can be generated. The threshold cycle of the unknown samples can then be quantified by reading off the standard curve.

1.600 1.400 1.200 1.000 0.800 0.600 0.400 0.200 0.000 -0.200

104 103

104 103

01 23456789 11 13 15 17 19 21 23 25 27 29 31 33 35 37 Cycle

40.00 35.00 30.00 25.00 20.00 15.00 10.00 0.00 -5.00

01 23456789 11 13 15 17 19 21 23 25 27 29 31 33 35 37 Cycle

Slope: -2.959 Y-Intercept: 34.644 Correlation 0.995 Coeff:

10 1 02 1 03 1 04 1 05 Starting quantity

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