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Limitations of Single-Strand Conformation Polymorphism Analysis As a High-Throughput Method for the Detection of EGFR Mutations in the Clinical Setting

The Ohio State University, Columbus, OH
The Ohio State University, Columbus, OH; Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom; Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH

To the Editor:

We read with interest the article by Marchetti et al¹ in the February 1, 2005, issue of the Journal of Clinical Oncology. The authors report on an analysis for epidermal growth factor receptor (EGFR) mutations in a panel of non–small-cell lung cancers (NSCLC) from 860 patients and concluded that the use of single-strand conformation polymorphism analysis (SSCP) was feasible for high-throughput scanning for EGFR mutations in NSCLC, presumably in the clinical setting. We would like to inject caution into their conclusion given the relative insensitivity of their recommended technology (SSCP) and the design of their experiments. Furthermore, such a conclusion may potentially bias our understanding of the correlation between EGFR mutation status and response to EGFR–tyrosine kinase inhibitors (TKI).

SSCP is probably the most commonly used conformation-based mutation scanning technology, because it is relatively simple, quick, inexpensive, and does not require expensive equipment. The reported accuracy, 60% to 90%, typically 65% to 70% for SSCP analysis, is highly dependent on several physical factors that need to be optimized for virtually each region of interest.^2,3 The detection sensitivity decreases further when amplicon sizes exceed 200 bp, typically smaller than each exon of EGFR. Therefore, as a mutation scanning technique, SSCP is more often used to analyze the known polymorphisms or hot-spot (identical) mutations at single loci, especially when used for clinical purposes, whether for diagnosis or prognostication.

With regard to the limitations of SSCP analysis outlined above, it needs to be questioned if at the current time we can rely solely on a very operator-dependent and gene-dependent technique. Several aspects of EGFR mutations and their role in carcinogenesis and/or response to small molecule therapy in various solid tumors are still under investigation. More importantly, no real "hot spots" have yet been identified. Other groups used high throughput techniques to screen for EGFR mutations in various tumors. One group⁴ used SSCP analysis similar to the method employed by Marchetti et al to screen more than 90 patients with breast adenocarcinoma and identified only one patient whose tumor harbored a mutation. This is in contrast to unreported findings by our group. Using direct sequencing, we were able to identify EGFR mutations in invasive breast adenocarcinomas from 6% of all patients. Furthermore, several of the point mutations identified were located within exon 20, an exon which was excluded from the Marchetti et al analyses. When SSCP was compared with three other rapid mutation scanning technologies in a blinded fashion to detect BRCA1 mutations and variants, only 60% of the known variations were found and reported.³ In this setting, denaturing high performance liquid chromatography (dHPLC) had a sensitivity of 100%.

We noted that the authors also employed heteroduplex analysis that is often carried out as an adjunct to SSCP analysis. Heteroduplexing is very sensitive in detecting mutations, even when the mutant contribution is as low as 1% to 10%. This very strength of heteroduplex analysis, the high sensitivity of detecting tiny mutant contributions, may very well mislead investigators and clinicians in the context of EGFR-TKI. The somatic EGFR-TKI mutations are heterozygous, ie, present on one of the two DNA strands, and so the mutant contribution of a pure tumor comprising all cells with the mutation would be 50%. Often, neoplasias are contaminated with a greater or lesser amount of normal surrounding tissue, thus decreasing the proportion of mutant DNA strands compared with wildtype. It is equally plausible that even within a tumor, only a population of clones harbor the mutation with remaining clones being wildtype. Direct sequencing can easily detect mutant contributions as low as the 25% to 30% range, while heteroduplex analysis can detect mutant contributions as low as 1% to 10%. However, what are the implications of detecting EGFR mutations in 1% of all tumor cells in the context of EGFR-TKI?

We therefore would like to suggest that until our understanding of the EGFR mutation spectra is as advanced as it is for such genes as BRCA1, PTEN, and others, we need to caution against high-throughput, low-sensitivity techniques in the setting where hot-spot mutations do not exist. This is particularly germane when such analyses are for clinical purposes to alter management. Perhaps the investigators studying EGFR somatic mutations in various neoplasias for prediction of response to small molecule inhibitors should come together in a consortium, similar to that done for BRCA1/2 (Steering Committee of the Breast Cancer Information Core),³ to study related questions.

Authors’ Disclosures of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.

Acknowledgment

C.E. is a recipient of the Doris Duke Distinguished Clinical Scientist Award. The Ohio State University Comprehensive Cancer Center is a recipient of grant P30CA16058 from the National Cancer Institute.

REFERENCES

1. Marchetti A, Martella C, Felicioni L, et al: EGFR mutations in non–small-cell lung cancer: Analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 23:857-865, 2005[Abstract/Free Full Text]

2. Hayashi K, Yandell DW: How sensitive is PCR-SSCP? Hum Mutat 2:338-346, 1993[CrossRef][Medline]

3. Eng C, Brody LC, Wagner TM, et al: Interpreting epidemiological research: Blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1. J Med Genet 38:824-833, 2001[Abstract/Free Full Text]

4. Huang SF, Liu HP, Li LH, et al: High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 10:8195-8203, 2004[Abstract/Free Full Text]

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  Antonio Marchetti, Fabio Barassi, Lara Felicioni, Carla Martella, Simona Salvatore, Andrea Mezzetti, Franco Cuccurullo, Fiamma Buttitta, Antonio Chella, Pier P. Camplese, Teodorico Iarussi, Felice Mucilli, and Rocco Sacco
  JCO 2005 23: 5848-5849 [Full Text]

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