Molecular assessment of tumour stage and disease recurrence using PCR-based assays
Section snippets
Molecular staging techniques
Molecular staging relies on PCR or reverse-transcription–PCR (RT–PCR) to detect the presence of tumour-derived cells in lymph nodes, the systemic circulation or bone marrow. A typical amplification reaction requires very small amounts of biological material and is carried out in an automated instrument that takes the reaction through a series of different temperatures for varying amounts of time (Fig. 3). Each PCR cycle consists of denaturation, annealing and polymerization steps and,
Choice of tissues for molecular staging
The spread of tumours requires a number of distinct steps, all of which must be completed successfully before the development of a distant metastasis[7]. Because many of these might be regulated epigenetically[8], there are no universal genetic markers for metastasis. In addition, the development of metastatic disease also depends on the host response[9], and qualitative or quantitative differences in patients' immune systems can allow some to mount more effective immune responses to their
Application of PCR
DNA-targeted PCR can detect qualitative differences in gene structure, such as loss of heterozygosity, chromosome rearrangements or point mutations between malignant and normal cells (Fig. 3). It can detect micrometastases from tumours that are defined by their genetic alteration, such as many haematopoietic malignancies, or where a high proportion of cancers of a particular kind harbour the same mutation. DNA isolated from lymph nodes, bone marrow and peripheral blood can be analysed. A
Application of RT–PCR
By contrast with PCR, RT–PCR detects differences in gene expression and is used to target tissue-specific mRNA expression (Fig. 3). Differentiated cells express different subsets of mRNAs, and numerous tissue- and tumour-specific mRNAs have been proposed as markers for micrometastasis detection[26]. RT–PCR relies on the absence of their expression in target tissue such as lymph nodes, bone marrow and blood. Their detection is assumed to correlate with the presence of tumour cells in that tissue
Limitations
Like every new technology, PCR has built-in technical and biological limitations that must be recognized and addressed if it is to become a routine tool in clinical diagnostics.
Concluding remarks
Unquestionably, the use of molecular techniques in tumour staging merits further intensive investigation because conventional histopathology does not allow the accurate prognostic stratification of patients. However, the wave of recent enthusiasm for these methodologies needs to be tempered until optimal standardized protocols for each of the common cancers are identified. It is increasingly recognized that true tissue-specificity of tumour cell-derived gene expression is a rare phenomenon.
Glossary
Amplicon—DNA product generated by PCR with ends defined by forward and reverse primers.
Epigenetic—Phenotypic alterations caused by modifications to genes that do not alter the DNA sequence (e.g. CpG-dinucleotide methylation).
Minimal residual disease—Occult sub-microscopic tumour depositions that are present after curative surgery.
Nested PCR—Two-stage amplification reaction. Primers that complement regions of the first-stage amplification product are used to amplify a portion of the original PCR
The outstanding questions
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Do molecular methods yield improved staging information compared with conventional staging methods?
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What is the clinical relevance of circulating tumour cells?
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Is the detection of micrometastases in bone marrow or blood as significant as their detection in lymph nodes?
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Is the detection of DNA molecules carrying tumour-specific mutations in plasma as significant as detecting the presence of tumour cells?
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Can quantitative PCR protocols overcome the problems associated with illegitimate expression?
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