Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI) frequently occur together in tumor cells

Abstract

Background: Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.

Methodology/principal findings: We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.

Conclusions: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Figure 1. Mutant allele specific imbalance (MASI) and some earlier observations.

a) types of MASI. Three major types of MASI may occur. b) Complete MASI of KRAS gene as identified in 1991. We reported KRAS mutations in non-small-cell lung cancer (NSCLC) cell lines using restriction fragment length polymorphism (RFLP) method which can digest only wild type (WT) allele. We made this figure using modified methodologies from the original publication . Three out of four KRAS mutant NSCLC lines showed homozygous mutations (complete MASI) of KRAS codon 12. NT, no treatment of restriction enzyme; +, presence of treatment of restriction enzyme. c) Our first EGFR mutation (exon 19 deletion) showed that the mutant allele was in great excess compared to the WT allele. WT, wild type.

Figure 2. Homozygous mutations (complete MASI) of oncogenes are frequent.

Quantitation of mutant allele (mA) by direct sequencing (a and b). wA, wild type allele; WT, wild type; mA%, proportion of mutant allele. a) Calculation method of mA% in point mutations by sequencing eletcropherogram is shown. b) An example of accuracy of mutant allelic quantitation (mA%) by measurement of sequencing electropherogram (KRAS mutation: G12V, 35G<T; results of forward reading is shown). We performed similar experiments for 14 kinds of mutations of KRAS, BRAF, PIK3CA or EGFR genes and confirmed the accuracy of mA% by measurement of sequencing electropherograms. F, forward sequencing; R, reverse sequencing c) and d) Frequency of homozygous mutations of 11 well-described tumor related genes in 833 cancer cell lines collected at Cancer Genome Project, Welcome Trust Sanger Institute (www.sanger.ac.uk/). As expected, homozygous mutations are frequent in six tumor suppressor genes (c). Those of five oncogenes are also relatively frequent (d). MASI, mutant allele specific imbalance; The prefix m- means mutant.

Figure 3. The association between mutations, copy number gain (CNG) and mutant allele specific imbalance (MASI) of EGFR pathway genes.

a) The association between mutations and CNG of EGFR pathway genes in cell lines and tumors across organs. We combined the data of cell lines and tumors because of similarity of both data sets. Mutations are more frequent than CNG in KRAS gene while CNG are more frequent than mutations in other genes. CNG are significantly correlated with mutations in KRAS (P<0.0001) and EGFR (P<0.0001) genes (*). However, mutations or CNGs of BRAF and PIK3CA genes are usually exclusive and rarely present together. b) The association between percent of mutant allele (mA%) and copy number for 75 mutations in 68 mutant cell lines. Gray dotted line is the hypothetical curve of mutant allele specific amplification. There were 36 mutations with MASI (48%), 38 with balanced (51%) and one with reverse MASI (1%). Thirteen mutant cell lines including mutant KRAS (n = 12) and BRAF (n = 1) had uniparental disomy (complete MASI without CNG) and four lines (all mutant KRAS) had complete MASI with modest level of CNG (copy number<9, black dotted circle). The prefix m- means mutant; mA%, proportion of mutant allele.

Figure 4. KRAS mutant allele specific imbalance (MASI) in lung cancer cell lines.

(Left upper) Copy number and allelic imbalance status as determined by SNP 6.0 arrays are depicted for representative cell lines with balanced and MASI patterns of KRAS mutant/wild type allele ratios. For copy number, each blue dot represents an array element ordered by genomic position. Those shifted to the left of the middle line have decreased copy number whereas those shifted to the right have increased copy number. For allelic imbalance, dashed lines represent regions with no imbalance whereas solid lines represent those with imbalance. Thicker solid lines represent the region of maximum imbalance across the chromosome arm (see methods). The genomic location of KRAS is indicated by the horizontal black line. (Left lower) Electropherograms of direct DNA sequencing with mutant allele proportion (mA%, determined by electropherogram) and KRAS copy number (copy#, determined by quantitative PCR) are present in the same cell lines used for SNP arrays. (Right lower) KRAS FISH in HCC1171 was performed using purified DNA from BAC clone RP11-1119I8 encompassing the KRAS gene (red signal) and CEP12-SpectrumGreen (Abbott Molecular, IL) as an internal control. Means of KRAS copy number are 21.6±11.0 (standard deviation, SD) and those of CEP12 number are 3.7±1.2 (SD). Both SNP arrays and gene specific assays confirm that HCC2347 displays neutral KRAS copy number with no imbalance (mutant/wild type balanced) whereas HCC1171 and H2030 display imbalance (MASI) with copy number gain (CNG) or uniparental disomy (UPD), respectively.

Figure 5. Different frequencies and mechanisms of MASI of EGFR pathway genes.

MASI is equally frequent in mutant KRAS and EGFR genes than others and PIK3CA MASI is rare (a). KRAS MASI is caused almost equally by uniparental disomy or copy number gain (CNG) while EGFR MASI is mainly caused by CNG (b). The prefix m- means mutant. MASI, mutant allele specific imbalance.

Figure 6. Biological role of mutant allele specific imbalance (MASI).

Gene dosage is highly associated with mRNA expression level (a). Proportion of mutant allele (mA%) determined by DNA sequencing electropherogram is significantly consistent with mA% by cDNA sequencing using different sets of primers (b). c) KRAS alterations are related to ras GTPase activity. KRAS mutations or copy number gains (CNGs) alone are related to high ras GTPase activity and the two molecular changes are synergistic. The prefix m- means mutant. HBEC, human bronchial epithelial cell; WT, wild type; UPD, uniparental disomy; *, KRAS mutation with CNG versus Others; **, KRAS mutation with CNG versus either KRAS mutation or CNG; ***, either KRAS mutation or CNG versus WT.

Figure 7. The effect of KRAS mutations and copy number gain (CNG) on clinical outcome in 237 lung adenocarcinomas.

The effect of 1) KRAS mutations (without CNG), 2) KRAS CNG (without mutations), 3) both KRAS mutations and CNG, and 4) others (without KRAS mutations and CNGs) on clinical outcome is shown. Tumors having both alterations indicate worse prognosis with borderline significance than all others (P = 0.04). The prefix m- means mutant.