In Reply:
Authors: 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, Rocco Sacco
Journal: Journal of Clinical Oncology (2005)

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 group 4 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.