EGFR mutation testing in pulmonary adenocarcinoma. Evaluation of tumor cell number and tumor percent in paraffin sections of 120 small biopsies

OBJECTIVES: Successful evaluation of EGFR mutational status in small biopsies may be hampered by the number of tumor cells present in the tissue section. The aim of the present study was to determine the minimal number of tumor cells necessary for a reliable EGFR mutation testing in lung adenocarcinoma. MATERIALS AND METHODS: The minimal number of tumor cells was determined experimentally in surgical specimens of 12 EGFR-mutated cases. DNA was extracted from progressively smaller tumor areas obtained with laser capture microdissection and was tested using a real-time PCR technique. The results were then validated in tissue sections of 120 small biopsies, where total number of tumor cells and percent tumor cells were determined in H&E digitalized slides. RESULTS: The laser capture microdissection study revealed that a tumor area of 0.12 mm(2), containing 140 ± 34 tumor cells, was large enough to allow detection of EGFR mutations in 11 of 12 cases. Moreover, it was also demonstrated that EGFR gene amplification and/or chromosomal polysomy could cause a 2-4-fold increase in the sensitivity of the assay. The reliability of these findings was tested in 120 small biopsies containing 26 EGFR-mutated cases. It was found that only a single case had <200 tumor cells, that the EGFR-mutated case with the lowest tumor content had 364 tumor cells occupying a tumor area of 0.12 mm(2), and that 11 of the 26 EGFR-mutated cases (42%) had <20% tumor cells. Finally, the incidence of EGFR-mutated cases in 145 small biopsies (21.4%) was similar to that detected in 132 surgical specimens (20.5%). CONCLUSIONS: Our findings suggest that when a small biopsy contains enough tumor cells to allow a histological diagnosis of adenocarcinoma it probably contains also an adequate number of tumor cells for a successful EGFR mutation testing if a real-time PCR technique is used.