Supplementary Material
Results
Clinical Features of 78 Analyzed Patients: doublets are associated with a later age of
onset.
When the 25 patients with EGFR mutations are compared with the 53 patients without
mutations, the average ages are similar (58.96 ± 14.96 vs. 61.43 ± 15.38 years).
Mutations are more frequent in females with lung cancer than in males (36% vs. 26%).
Mutations are more frequent in non-smokers than in smokers (61.9% vs.38.1%). Within
the constraint of small sample size, doublets appear more frequently in older people
(average age 72.3 ± 17.4 compared with 56.2 ± 14.1 for singlets).
Unamplified vs. Amplified EGFR Alleles
Microdissection is generally performed in regions in which the overwhelming majority of
cells are malignant by histological analysis. Of the 25 EGFR somatic mutations, 8
displayed MAD (mutant allele dominant chromatogram), and 2 were putatively
hemizygous, reflecting dramatic amplification of the mutant allele, although loss of
heterozygosity cannot be ruled out. All exons of EGFR were sequenced in the 8 samples
with MAD. Except in one case (possibly contaminated by normal cells), all germline
polymorphisms in the entire EGFR gene displayed a MAD pattern. This suggests that the
MAD pattern is not merely a sequencing artifact, but that amplification of the entire
mutated EGFR gene has occurred. Curiously, one of the samples showed a MAD pattern
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in the kinase region, but multiple polymorphisms in the remainder of the gene were
homozygous. None of the tumors with EGFR mutations showed a wild type dominant
mutation chromatogram.
Twenty tumors without an EGFR mutation in the kinase domain were sequenced for the
remaining exons. All the polymorphisms were balanced (~50:50) except one case which
contains amplified EGFR wild-type allele. Thus, 32% of the somatic EGFR mutations
were associated with amplification of the mutant allele, none of the EGFR mutations
showed a wild-type allele dominant chromatogram, and 5% of the tumors without EGFR
mutations contained an amplified EGFR allele.
Discussion
Are EGFR mutations associated with gene amplification in lung cancers?
EGFR gene amplification and EGFR mutations are common in lung cancer. The
relationship between the two events is not well characterized. Gene amplification of
mutated alleles produce a mutant allele dominant chromatogram pattern (32% of total
mutations in our cohort), consistent with selective amplification of the mutant allele (Ma
et al. 2007; Shigematsu et al. 2005; Takano et al. 2005). Four of six non-small cell lung
cancer cell lines with EGFR mutations also have EGFR amplification (Okabe et al.
2007). In human glioblastomas, EGFR mutations are frequently associated with EGFR
gene amplification (Frederick et al. 2000). If alleles with EGFR mutations are amplified,
the response to TK inhibitors may differ relative to mutant alleles without gene
amplification. EGFR amplification has been identified as an alternative predictor of
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response to TK inhibitors and is associated with elevated EGFR gene transcript and
protein levels (Cappuzzo et al. 2005; Tsao et al. 2005). Future coordinated analysis of
gene mutations, amplifications of normal and mutated genes, and expression of normal
and mutated genes may improve prediction of drug response.
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Legends to Figures
Fig S-1: EGFR mutation chromatogram patterns: Balanced heterozygote (BH), mutantallele-dominant pattern (MAD), mutant-allele-only (MAO). Mutations in chromatograms
1-6 are: (1) c.2235_2249del, (2) c.2240_2257del, (3) c.2235_2249del, (4) c.2573T>G, (5)
c.2573T>G, (6) c.2573T>G.
Fig S-2: Three doublets are composed of in cis mutations.
Sequences are shown from the original tissue, from the cloned wild type allele, and from
the cloned mutant allele for each of the three doublets. Set A and set C show the upstream
sequences. Set B shows the downstream sequence. Each allele was cloned for two of the
doublets (A, B). The third doublet (C) was amplified by allele-specific primers: one wild
type allele specific primer (A) or one mutant allele specific primer (T) plus a primer
flanking the second mutation. Sequencing of the amplified product indicates that the
mutations are allelic. Pink arrows point to mutated sites.
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