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Letters to the Editor |
Laboratory for, Experimental Oncology, Department of, General Medical Oncology, University Hospital Gasthuisberg, Onderwijs & Navorsing 1, Room 815, Herestraat 49, 3000 Leuven, Belgium, Fax 32-1634-6901, E-mail julie.decock{at}med.kuleuven.be
To the Editor:
I read with great interest the recent article by Li et al. (1), in which they presented a novel, cost-effective quantitative PCR technology for the analysis of clinical cancer samples. They developed an antiprimer quenching-based real-time PCR (aQRT-PCR) that uses fluorescently labeled PCR primers in combination with a universal quenching antiprimer, reducing the cost of labeling. In agreement with their in-house fluorescence in situ hybridization (FISH) and immunohistochemistry results, their multiplex aQRT-PCR approach detected chromosomal Her2/neu amplification in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA. Hence, Li et al. (1) concluded that aQRT-PCR could be used as a simple, versatile, reliable, and low-cost alternative for Her2/neu detection in clinical cancer samples.
Her2/neu overexpression is considered a strong prognostic and predictive marker for breast cancer. Overexpression of Her2/neu is seen in 
30% of invasive human breast carcinomas. Because of their low cost and ease of performance, immunohistochemical methods are commonly used to measure Her2/neu protein. In 90% of breast carcinomas, Her2/neu protein overexpression is attributable to gene amplification. Although FISH measurement of Her2/neu gene amplification is considered the gold standard for clinical detection of Her2/neu, FISH analysis is more time-consuming and expensive than immunohistochemical methods and is therefore not preferred for primary screening of Her2/neu status.
Quantitative real-time PCR using paraffin-embedded tissue is a suggested alternative standardized approach to Her2/neu detection (2)(3)(4), but I think that the multiplex aQRT-PCR for Her2/neu detection, as presented by Li et al. (1), should be used very cautiously in clinical cancer samples. HER2/neu overexpression has been linked with polysomy of chromosome 17. Polysomy 17 has been reported in breast carcinoma cases with borderline or low Her2/neu protein concentrations in the absence of gene amplification (5)(6). Thus, for reliable quantification of Her2/neu gene amplification, an internal control for chromosome 17 should be used. The use of a probe directed against the centromere of chromosome 17, as in dual-color FISH, can differentiate between true Her2/neu gene amplification and polysomy of chromosome 17. Because Li et al. (1) used glyceraldehyde-3-phosphate dehydrogenase (GAPDH), located on chromosome 12, as an internal control, centromere involvement could not be distinguished from true gene amplification. Moreover, because no details on the FISH procedure were reported, the comparison of the aQRT-PCR with their in-house FISH results might be questioned. From the data in the article, it is not clear whether a single-color (Her2/neu) or dual-color (Her2/neu probe–centromere 17 probe) FISH protocol was used.
I understand that the main point of the report by Li et al. (1) was the development and validation of the accuracy of the aQRT-PCR methodology, and I think that this method has great potential for analysis of clinical cancer samples. However, it requires revision for Her2/neu detection. I hope to see optimization of the aQRT-PCR method for Her2/neu detection in clinical cancer samples in future work by Li et al., as this would be of great value for clinical cancer research.
Acknowledgments
J. Decock is a research fellow supported by a grant from the Vlaamse liga tegen kanker and the EU Framework Programme 6 project LSHC-CT-2003-503297 (cancerdegradome).
References
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