Frequency and type of epidermal growth factor receptor mutations in African Americans with non-small cell lung cancer

Abstract

Background

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer predict response to tyrosine kinase inhibitors (TKIs). Mutations occur more commonly in never-smokers and East Asians but there are conflicting reports on the frequency of EGFR mutations in tumors from African Americans.

Methods

Tumors from 67 African American and 77 Caucasian participants in previous case-control studies of lung cancer were selected to determine EGFR mutational status. Mutation analysis was performed using the Sequenom mass array analyzer (Sequenom, San Diego, CA).

Results

Overall, 13.9% of the study population carried an EGFR mutation. EGFR mutations occurred in 11.9% of tumors from African Americans compared with 15.6% in Caucasians (p=0.53). All mutations found in African Americans were deletions in exon 19. The majority of mutations were found in non-smokers among both African Americans (7/8) and Caucasians (8/12).

Conclusion

These results indicate that African Americans with non-small cell lung cancer (NSCLC) harbor somatic EGFR mutations at a frequency similar to Caucasians with NSCLC. Thus, clinicians should not use race as a clinical decision parameter for the use of EGFR-TKIs.

Introduction

Epidermal growth factor receptor (EGFR) is a tyrosine kinase involved in cell signaling and somatic mutations in patients with non-small cell lung cancer (NSCLC) are predictive of response to EGFR tyrosine kinase inhibitors (TKIs), with 70–80% of patients deriving substantial benefit from this therapy. ^{1, 2} Several studies have documented that most patients responding to EGFR-TKIs have mutations in the ATP-binding regions of the gene.^3–6 More than 90% of these mutations involve either a deletion in exon 19 or a point mutation (L858R) in exon 21.⁷

EGFR mutations occur predominantly in never-smokers and are more common in patients with adenocarcinoma, East Asian ethnicity and in females.^3–6 Less is known about the mutation frequency in other ethnic groups, and studies in African American patients have yielded conflicting results. Two studies reported that the EGFR mutation frequency in African Americans was about 2% compared with the reported frequency of 15–20% in whites.^7,8 In contrast, Riely et al.⁹ reported 42.9% of the 14 tumors they studied from African Americans harbored EGFR mutations.

The decision to offer EGFR testing is usually determined by whether the patient matches the clinical profile of an individual who would be likely to harbor a mutation. The purpose of this investigation was to examine the frequency and characteristics of EGFR mutations in a cohort of African Americans and whites with NSCLC to determine whether a difference in EGFR mutation frequency exists by race.

Methods

Tumors from participants in previous population-based case-control studies of lung cancer from 1985–2008 (A.G.S.) who had consented to allow their tissue to be used for research purposes were selected to determine EGFR mutation status. These subjects had detailed demographic and information (age, race, smoking status, pack years of smoking) available from previous interviews, and clinical information (histology, stage, and grade) available from the Metropolitan Detroit Cancer Surveillance System, part of the national Surveillance, Epidemiology and End Results (SEER) program. Never smokers, light smokers and individuals with adenocarcinoma were oversampled, and cases were frequency-matched by race on sex, smoking status, and histology. Never smokers were defined as individuals smoking fewer than 100 cigarettes in their lifetime.

We employed the OncoCarta Panel V1.0 developed by Sequenom, which examined 238 mutations in 19 different oncogenes. This method was chosen because of its sensitivity (as low as 10% mutant allele frequency) and high throughput. For the purposes of this analysis, we focused on 43 known mutations in EGFR included in this panel. Mutation analysis was performed using the mass-spectroscopy based MassArray device developed by Sequenom. DNA was extracted from formalin-fixed paraffin embedded tumors using a standard kit (Qiagene, 51306). Briefly, an initial PCR reaction is performed to amplify a small region which included the potential point mutation site. Next, a 10 base DNA oligonucleotide primer binds immediately upstream of the mutation site and is extended by one base into the potential mutation site. The primers are subsequently separated on a matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer which is able to quantitatively discern the specific nucleotide that was extended.

Differences by race and mutation status were assessed for categorical variables using p-values from chi-squared tests or Fisher’s exact test when expected cell frequency was less than 5. Comparisons were made using t-tests for continuous variables. Differences were considered to be statistically significant at alpha<0.05. All analyses were performed using SPSS Version 18.

Results

Tumor samples were obtained from 67 African American and 77 white patients with NSCLC. The demographic characteristics of the patients are listed in Table 1. At the time of diagnosis, African Americans were older compared to whites (p=0.03) but other key parameters did not differ between racial groups. EGFR mutations were found in 13.9% of tumors from the total population (Table 2). 15 mutations occurred in never-smokers with an additional 2 mutations found in patients with a smoking history of only 1 pack year. The majority of mutations were found in adenocarcinomas (n=18), with 2 squamous cell carcinomas also harboring a mutation (p=0.10). The mutation frequency in never smokers was 47% (data not shown). EGFR mutations did not differ by race with a frequency of 11.9% in African Americans and 15.6% in whites (p=0.53). Figure 1 illustrates the relationship between smoking status, race and frequency of different mutation types. Mutations were found in seven of 11 tumors from African American never-smokers (63.6%) and 8 of 21 (38.1%) tumors from white never-smokers (p-value=0.27). All 8 mutations in African Americans were exon 19 deletions whereas mutations in whites included exon 19 deletions (n=7, 58.3%), the L858R point mutation in exon 21 (n=3, 25%), and two exon 18 mutations, E709A and G719S (16.7%). There was no statistically significant difference between the types of mutations carried by race (p-value=0.17, data not shown).

Table 1.

Demographic characteristics of the study population, by race

	Whites (n=77)	African Americans (n=67)	p-value
	N (%)	N (%)
Mean age at diagnosis	55.2	60.0	0.03
Sex			0.20
Male	19 (24.7)	23 (34.3)
Female	58 (73.3)	44 (65.6)
Smoking status			0.12
Never	21 (27.3)	11 (16.4)
Ever	56 (72.7)	56 (83.6)
Histology			0.75
Adenocarcinoma	57 (74.0)	48 (71.6)
Other NSCLC	20 (26.0)	19 (28.4)
Stage at diagnosis			0.47
I	30 (39.0)	20 (29.9)
II	31 (40.3)	29 (43.3)
III	13 (16.9)	15 (22.4)
Missing	3 (3.8)	3 (4.4)

Table 2.

Patient characteristics by EGFR mutation status

	EGFR Mutations (n=20)	Wild Type (n=124)	p-value
	N (%)	N (%)
Ethnicity			0.53
White	12 (60.0)	65 (52.4)
African American	8 (40.0)	59 (47.6)
Mean age at diagnosis	63.8	56.4	0.02
Sex			0.33
Male	4 (20.0)	38 (30.6)
Female	16 (80.0)	86 (69.4)
Smoking status			<0.001
Never	15 (75.0)	17 (13.7)
Ever	5 (25.0)	107 (86.3)
Histology			0.10
Adenocarcinoma	18 (90.0)	87 (70.2)
Other NSCLC	2 (10.0)	37 (29.8)
Stage at diagnosis			0.18
1	3 (15.0)	47 (37.9)
II	10 (50.0)	50 (40.3)
III	5 (25.0)	23 (18.5)
Missing	2 (10.0)	4 (3.2)

Figure 1.

Number of EGFR mutations by race and smoking status.

Discussion

Lung cancer remains the leading cause of cancer-related death in the United States with estimates of more than 150,000 deaths in 2009, and an overall 5-year survival of 16%.¹⁰ The majority of these cases are NSCLC. EGFR-TKIs have extended survival in some NSCLC patients. Clinical and biological studies have determined that TKI-responders are primarily those with EGFR mutations.^3–6 The recently completed Phase III IPASS trial reported that the hazard ratio for progression or death was 0.48 in patients with EGFR mutations treated with gefitinib compared with those treated with carboplatin-paclitaxel (p<0.001).¹ The 12-month progression-free survival rate was significantly higher with gefitinib in that study of nonsmokers or former light smokers with adenocarcinoma (24.9% in the gefitinib group vs. 6.7% for carboplatin-paclitaxel).¹ This study illustrates the clinical relevance of the EGFR mutational status for therapeutic decision making in patients with advanced NSCLC.

Contrary to recent reports, we observed no statistically significant difference in EGFR mutation frequency in tumor samples from whites and African Americans, in the largest study of African Americans to date. Studies reporting the frequency of EGFR mutations, including the present report, must be interpreted with caution since the composition of the patient population may influence the results. EGFR mutations occur predominantly, although not exclusively, in never-smokers. Pao et al.⁵ found EGFR mutations in only 4 of 481 samples from smokers compared with 7 mutations in 15 tumors from never-smokers. This mutation rate is similar to our observation of 15 mutations in 32 never-smokers. Since approximately 10% of patients with NSCLC are never-smokers, EGFR mutation rates will be low unless the study population is enriched with these individuals.

The mutation frequency in whites in our study is similar to other reports in white populations.^7–9 In African Americans, Riely et al.⁹ studied 14 tumors (8 were never smokers) and found EGFR mutations in 6 patients, similar to our findings that approximately 2/3 of African American never smokers harbored a mutation in EGFR. It is unclear why Yang et al.⁷ and Leidner et al.⁸ both reported finding only a single EGFR mutation after studying 41 and 53 African Americans with NSCLC respectively, but it may have been due to the inclusion of relatively few never smokers in these investigations, or chance.

EGFR mutations of importance in NSCLC appear to be confined to the first four exons (18–21) of the ATP-binding region of the tyrosine kinase receptor. In our white subjects, exon 19 mutations accounted for two-thirds of EGFR mutations, yet all of the mutations observed in African Americans were deletions in exon 19. Larger studies are needed to determine whether significant differences exist in mutation type between African Americans and whites or whether this is an anomaly related to the sample size or some unique characteristic of our study cohort.

Determination of mutation type may have clinical relevance since some studies have demonstrated that treatment response varies with mutation type. Riely et al.⁹ reported that patients with exon 19 deletions treated with TKIs had a median survival of 34 months compared with only 8 months in those with the exon 21 L858R point mutation. Jackman et al.¹¹ found that length of survival was more than doubled (38 months vs. 17 months) for patients with exon 19 deletions. In addition, a study examining the association between EGFR mutations and outcome of erlotinib treatment found a higher probability of response associated with the exon 19 deletion (p-value=0.001).¹² These findings have not been confirmed in two studies involving Japanese patients where no significant difference was noted in progression-free survival based on EGFR mutation type.^13,14

In conclusion, this study suggests that EGFR mutations occur as frequently in African Americans as in whites. Thus, clinicians should not use race as a clinical decision parameter for the use of EGFR-TKIs.