The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)