Reverse Transcription Polymerase Chain Reaction

In molecular methods to detect MRD, total RNA is first isolated from mononuclear cells. mRNA is reverse-transcribed to cDNA, then amplified by PCR using target gene-specific primers. In some assays, nested PCR is carried out, i.e., an aliquot of the PCR product is subjected to a second PCR reaction, although carryover contamination is a concern. The target gene is identified by the presence or absence of a band of the appropriate molecular size after separation by agarose gel electrophoresis of the PCR product. In some cases, the presence of a gene transcript is further verified using Southern blot, as well as DNA sequencing.

11.7.3.3.1 Real-Time Quantitative RT-PCR

The emergency of real-time quantitative PCR (qRT-PCR) technology permits not just the identification of a target gene, but also its transcript level (Heid et al. 1996; Gibson et al. 1996). Such a development has broadened the potentials in MRD monitoring. With a wide linear dynamic range, superior sensitivity and accuracy, real-time RT-PCR allows good intra-assay and inter-assay reproducibility. Additional attractions include high throughput capacity, speed, and elimination of lengthy post-PCR handling steps, preventing potential carryover contamination. Two major technical variables that need to be addressed are the selection of a reference gene against which the test samples can be normalized, and how to discriminate a positive from a negative result.

11.7.3.3.2 Molecular Targets

Several molecular targets have been studied extensively in the detection of residual NB cells in the BM and PB. Specificity is determined by the absence of gene expression when a series of normal BM and PB is evaluated. Sensitivity experiments are carried out by spiking varying concentration of tumor cells from a NB cell line to normal mononuclear cells, and assess the limit of detection. Depending on the specific gene expression of the cell line, sensitivity can reach as high as 1/107. Tyrosine hydroxylase (TH, tyrosine 3-monoxygenase), being the first and rate-limiting en zyme in the biosynthesis of catecholamine, is a logical choice since most NBs secrete catecholamines. Detection of occult NB cells by RT-PCR of TH mRNA was first reported in BM by Naito et al. (1991), and by Burchill et al. (1994) in PB. Recently, several real-time RT-PCR assays for TH transcript have also been developed (Träger et al. 2003; Tchirkov et al. 2003).

Another useful molecular target is the transcript of GD2/GM2 synthase (ß1,4-N-acetylgalactosaminyl-transferase). It is the key enzyme required for the synthesis of GD2 (Furukawa et al. 1996), an antigen ubiquitously expressed on NB. Its utility as a molecular marker in the detection of NB cells in the BM was first reported by Cheung et al. (Cheung and Cheung 2001; Lo Piccolo et al. 2001). A highly sensitive and specific quantitative RT-PCR assay which measures GD2 synthase mRNA was developed with transcript levels correlating well with the number of NB cells as measured by immunocytology (Cheung and Cheung 2001).

The cancer testis antigen GAGE belongs to a family of genes which encode distinct tumor-associated peptides recognizable by autologous cytolytic T lymphocytes when presented by HLA class-I molecules (Van den Eynde et al. 1995). It is expressed in human tumors of different histologic types including NB,but is silent in normal adult tissues except for placenta and testis (De Backer et al. 1999). GAGE was demonstrated to be a potentially useful MRD marker of NB (Cheung and Cheung 1997; Cheung et al. 2000) and melanoma (Cheung et al. 1999).

11.7.3.3.3 Perspectives on Molecular Detection

Molecular-based MRD assays can be hampered by the inherent pitfalls of DNA amplification. False-positive findings may result from "tumor-specific" genes which are occasionally transcribed even in normal tissues. Illegitimate transcription, i.e., the transcription of any gene in any cell type, and pseudogenes, which lack intronic sequences resulting in PCR products indistinguishable from those generated from the mRNA, also pose concerns. False-negative results in molecular assays can be due to degraded RNA,tumor cell heterogeneity, the presence of inhibitors, technical errors, sampling problem, as well as down-regulation of the target gene.

Table 11.7.2 MRD detection with prognostic significance in survival (p<0.05)

Study

Number of Patients

Sample

Time from diagnosis

Survival

Detection method

Reference

0 0

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