INTERNATIONAL JOURNAL OF CLINICAL INVESTIGATION AND CASE REPORTS - Volume 1, Issue 2, (Aug-Oct)

Pages: 45-51

Date of Publication: 09-Sep-2022

Print Article   Download XML  Download PDF

Epithelial Growth Factor Receptor Mutations Predict Hepatocellular Carcinoma Patient’s Prognosis

Author: Lo Wei-Ching , Jung-Sheng Chen , Wen-Chi Yang* (TAIWAN)

Category: Genetics

Abstract:

Objective: Hepatocellular Carcinoma (HCC) is an aggressive malignancy and the second leading cause of cancer-related death in the world. Epithelial Growth Factor Receptor (EGFR) overexpression has been demonstrated in HCC patients frequently and is a poor prognostic factor. EGFR is a promising target for cancer treatment, but its variety in response by mutation status.

Patients and Methods: We retrospective analyzed 28 Hepatocellular Carcinoma (HCC) patients with good quality DNA samples, 5 hepatic dysplasia tissues, and 5 cholangiocarcinoma samples from 1996 to 2002. EGFR mutation status was detected by ARMS and Scorpions real-time PCR, using therascreen^® EGFR RGQ PCR Kit. T790M, exon 19 deletions, L858R, L861Q, G719X, S786I, and exon 20 insertions can be detected. Clinical data of 28 HCC patients were collected for the longest period of 17 years.

Results: All patients received liver segmentectomy. No one received tyrosine kinase inhibitor treatment during the following period. All liver samples had T790M mutation detected. Based on overall survival, we calculated cut-off points, by ROC curve. In HCC patients with a higher percentage of T790M or G719X mutations had better overall survival (P=0.001, and 0.036). Patients with exon 19 deletion showed a trend of better overall survival. HCC patients with exon 19 deletions, L858R and G719X mutations showed trends of longer Disease Free Survival (DFS) and HCC patients with higher S768I mutation had a trend of shorter DFS. HCC samples from chronic hepatitis B carriers had a higher T790M mutation rate and the ones from chronic hepatitis C carriers showed the opposite in T790M mutation with statistics significant (P=0.013). No EGFR mutations contribute to HCC recurrence.

Conclusion: EGFR mutation status can predict HCC patient’s prognosis post liver segmentectomy.

Keywords: Hepatocellular Carcinoma , Cholangiocarcinoma, EGFR, T790M Mutation , G719X Mutation

DOI: 10.55828/ijcicr-12-09

DOI URL: http://dx.doi.org/10.55828/ijcicr-12-09

Full Text:

INTRODUCTION

Hepatocellular Carcinoma (HCC) affects people all over the world. Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer-related death worldwide and the seventh most prevalent cancer overall [1]. Asia and Africa have the highest incidence rates in the world [2]. The majority of the disease burden (85%) is carried by developing nations, with Hepatitis B Virus (HBV) infection being the most common there [3]. HCC occurrences are lower in Europe and the US than in Asia and Africa, although they have been steadily rising in recent years [4]. HCC treatment options include chemoembolization, Radiofrequency Ablation (RFA), systemic therapy with targeted medicines or chemotherapies, as well as potentially curative treatments (resection, transplantation, or local ablation). The recent positive Randomized Controlled Trial (RCT) of the multikinase inhibitors (MKIs) sorafenib and lenvatinib, which represents a breakthrough in the management of this neoplasm, has changed the absence of standard systemic therapy for advanced cases.

The molecular pathogenesis of HCC is very complex [5]. Epithelial Growth Factor Receptors (EGFRs) are widely expressed in human HCC, according to a number of studies [6-8], which likely contributes to the aggressive development characteristics of these tumors. A 170 kDa transmembrane tyrosine kinase receptor is known as the EGFR. For several cellular processes, including survival, proliferation, differentiation, and motility, canonical EGFR signaling is essential [9]. EGFR overexpression has been shown to be a poor prognostic factor, which was associated with extrahepatic metastases and early recurrence [7,10]. In HCC, EGFR expression is also significant, and pre-malignant liver nodules have EGFR overexpression [10]. In Non-Small Cell Lung Cancer (NSCLC) patients, EGFR exons 18-21 somatic mutations were found to be substantially connected with clinical response to tyrosine kinase inhibitors, gefitinib, and erlotinib treatment, as well as related to disease prognosis [11-13]. Exons 19-23 were shown to have missense and silent mutations in 13/33 (39.4%) and 11/33 (33.3%) of HCC tissues, respectively [14]. Here we analyzed EGFR mutation status in HCC patients, hepatic dysplasia, and a few cholangiocarcinoma patients and compared it with hepatitis B and C status. We also analyzed survival base on EGFR mutation status.

MATERIALS AND METHODS

Patients Samples Collection

This study is approved by Yuan’s General Hospital Institutional Review Board with IRB number 20140314B. We collected 48 HCC tissues from patients, from 1996 to 2002. However, only 28 patient samples can be used due to the good quality of DNA and related complete clinical data records. We also collected 5 hepatic dysplasia tissues and 5 cholangiocarcinoma tissues during the same period. We also choose 3 NSCLC without EGFR mutations as a control. The survival status was based on a retrospective chart review.

DNA Isolation

We cut the tumor part from our frozen tissue samples to isolate DNA. After being frozen in liquid nitrogen, the tumor part became light yellowish and harder than the normal part. DNA was isolated by using QIAamp^® DNA Mini and Blood Mini kit (QIAGEN^®, Hilden, GERMANY), from tissue.

Epithelial Growth Factor Receptor Mutations Detection

Epithelial Growth Factor Receptor (EGFR) mutations were analyzed with therascreen^® EGFR RGQ PCR Kit (QIAGEN^®, Hilden, GERMANY). The EGFR mutations are included point mutation (T790M, L858R, L861Q, S768I, and G719X), Exon 19 deletion, and exon 20 insertion. The protocol was followed by therascreen® EGFR RGQ PCR Kit Instructions for Use handbook. We keep the internal control cycle threshold (Ct) ratio between 18-22.

Statistics

We used the Receiver Operating Characteristic (ROC) curve to estimate the cutoff point for EGFR mutation to predict death in HCC patients. We analyzed the patient's overall survival and disease-free survival according to EGFR mutation status, using the Kaplan-Meier survival curve. ANOVA was used to analyze chronic hepatitis B, chronic hepatitis C, HCC cell differentiation status, liver cirrhosis, recurrent rate, and EGFR mutation status. ANOVA was also used to analyze EGFR mutation status and sex between HCC, dysplasia, and cholangiocarcinoma patients.

RESULTS

Patient’s Characteristics

We collected 28 Hepatocellular Carcinoma (HCC) samples, 5 hepatic dysplasia tissues, and 5 cholangiocarcinoma samples from 1996 to 2002 and detected EGFR mutation status by ARMS and Scorpions real-time PCR, using therascreen^® EGFR RGQ PCR Kit. T790M, exon 19 deletions, L858R, L861Q, G719X, S786I, and exon 20 insertions can be detected.

All patients were operable and received liver segmentectomy. The patients’ characteristics showed in Table 1. There is no significant difference of ages and sex between HCC, dysplasia, and cholangiocarcinoma. According to the cut-off value suggestion in therascreen^® EGFR RGQ PCR Kit Instructions, all tumors were not defined as EGFR mutation or exon 19 deletion. The cut-off values of T790M, exon 19 deletion, L858R, L861Q, G719X, and S786I are 7.4, 8.0, 8.9, 8.9, 8.9, and 8.9, separately. However, all liver samples had T790M and L858R mutations detected. We used the Receiver Operating Characteristic (ROC) curve to estimate the cutoff point for EGFR mutation to predict death in HCC patients. Based on ROC analysis, we found the cut-off point in T790MdCt of 14.035, deletion19dCt of 23.84, L858RdCt of 19.015, and G719X of 16.09, to define mutation status. For L861Q and S786I, we defined mutation if they can be detected in therascreen® EGFR RGQ PCR assay. Less HCC patients had L858R and L861Q mutations, compare with dysplasia and cholangiocarcinoma patients, with statistical significance (L858R, P=0.033; L861Q, P=0.025). Other EGFR mutations, including T790M mutation, deletion 19, G719X, and S768I mutations were similar between hepatocytes dysplasia, HCC, and cholangiocarcinoma. No exon 20 insertions were detected in our samples.

Hepatitis B/C and EGFR Mutations

We analyzed if EGFR mutations are related to hepatitis B or hepatitis C infection status. Only T790M mutation status is related to hepatitis B and hepatitis C carriers. More than 85% of hepatitis B carrier patients and only 25% of hepatitis C carrier ones showed T790M mutation status (Table 2). However, only hepatitis C carrier patients had a negative correlation with T790M mutation, with statistics significant (Table 3). There were no significant differences in EGFR mutation status between patients with liver cirrhosis or not, and different differentiation statuses of HCCs.

HCC Recurrent was Not Related to EGFR Mutation Status

In our patient's cohort, eleven patients in 28 HCC patients had recurrent under the follow-up period. However, there was no significant difference in EGFR mutation status (Table 4). There were no significant differences in EGFR mutation between cell differentiation statuses (Table 5). They were not related to liver cirrhosis in our patients’ cohort.

EGFR Mutation and Survival

The longest follow-up period in our cohort was 17 years. Based on ROC analysis, we found the cut-off point in T790MdCt of 14.035, deletion19dCt of 23.84, L858RdCt of 19.015, and G719X of 16.09, to defined mutation status. In HCC patients with higher expression of T790M mutation had better overall survival (median OS, mutation vs nonmutation: 5165 days vs 378 days; P=0.001), as well as patients with higher G719X mutation (median OS, mutation vs nonmutation: not reach vs 1308 days; P=0.036). And patients with exon 19 deletion showed a trend of better overall survival (median OS, mutation vs nonmutation: 5165 days vs 554 days; P=0.106). HCC patients with exon 19 deletions, L858R, and G719X mutations showed trends of longer disease-free survival (DFS) (median DFS, mutation vs nonmutation: 4499 days vs 75 days; P=0.001; 4499 days vs 798.677 days, P=155; 4499 days vs 798 days, P=0.113, separately). On the other hand, HCC patients with S768I mutation had a trend of shorter DFS (median DFS, mutation vs nonmutation: 291 days vs not reach; P= 0.199) (Figure 1).

Table 1: Patients’ characteristics.

	HCC	Dysplasia	Cholangiocarcinoma	P value
	(n=28)	(n=5)	(n=5)
Sex (M/F)	23/5	4-Jan	2-Mar	0.037#
Age	58 (18-82)	43 (29-54)	66 (50-74)	0.047#
Differentiation
Well	2
Moderate	13
Poor	5
Undefined	8
EGFR mutations
T790M#(%)	14 (50%)	1 (20%)	4 (80%)	0.259
Exon19 deletion$ (%)	22 (78.57%)	4 (80%)	3 (60%)	0.124
L858R& (%)	17 (60.71%)	4 (80%)	4 (80%)	0.033*
L861Q (%)	2 (7.14%)	1 (20%)	3 (60%)	0.025*
G719X@ (%)	14 (50%)	4 (80%)	2 (40%)	0.461
S768I (%)	7 (25%)	0	0	0.151
CR (%)	13 (46.43%)
Recurrent (%)	11 (84.6%)
# T790M Q-RT-PCR cut off value: 14.035; $ Exon 19 deletion Q-RT-PCR cut off value: 23.84; & L858R Q-RT-PCR cut off value: 19.015; @ G719X Q-RT-PCR cut off value: 16.09; * P<0.05

Table 2: EGFR mutation status and HBV, HCV status.

	Hepatitis B carrier	P value	hepatitis C carrier	P value
T790M# (mut)	85.71%	0.032*	25%	0.012*
Exon 19 deletion$(mut)	85.71%	0.557	100%	0.306
L858R&(mut)	57.14%	0.705	50%	0.716
L861Q(mut)	0	0.784	25%	0.51
G719X@(mut)	100%	0.757	75%	0.386
S768I(mut)	14.29%	0.894	25%	0.921
# T790M Q-RT-PCR cut off value: 14.035; $ Exon 19 deletion Q-RT-PCR cut off value: 23.84; & L858R Q-RT-PCR cut off value: 19.015; @ G719X Q-RT-PCR cut off value: 16.09; * P<0.05

Table 3: EGFR mutation and HBV, HCV infection.

	HBsAg		P value	Hepatitis C carrier		P value
	Positive (7)	Negarive (7)		Positive (4)	Negarive (10)
T790MdCt (median, 95% CI)	13.43 (13.245-13.812)	14 (13.297-14.291)	0.278	14.115 (13.604-14.586)	13.4 (13.226-13.75)	0.013*
Exon 19 deletion dCt(median, 95% CI)	18.725 (17.879-23.318)	19.91 (17.529-20.014)	0.184	19.825 (15.093-26.042)	18.75 (17.5897-20.892)	0.442
L858RdCt (median, 95% CI)	18.99 (17.574-19.309)	19.62 (17.718-28.237)	0.205	18.615 (15.652-22.743)	19.09 (17.903-19.931)	0.78
L861QdCt (median, 95% CI)	Not detected	22.77	0.337	22.77 (-12.42-23.80)	Not detected	0.117
G719XdCt (median, 95% CI)	15.84 (15.373-17.892)	16.155 (14.912-17.185)	0.416	16.32 (13.019-19.022)	15.91 (15.551-17.381)	0.603
S768IdCt (median, 95% CI)	20.43	23.12 (-35.71-81.952)	0.794	27.75	19.46 (7.135-31.785)	0.127
# T790M Q-RT-PCR cut off value: 14.035; $ Exon 19 deletion Q-RT-PCR cut off value: 23.84; & L858R Q-RT-PCR cut off value: 19.015; @ G719X Q-RT-PCR cut off value: 16.09; * P<0.05

Table 4: Recurrent and EGFR mutations.

	Liver cirrhosis	P value	Recurrent	P value
	(n=16)		(n=11)
T790M#(%)	9 (56.25%)	0.432	7 (63.64%)	0.137
Exon19 deletion$ (%)	13 (81.25%)	0.899	9 (81.82%)	0.844
L858R& (%)	9 (56.25%)	0.391	6 (54.55%)	0.826
L861Q (%)	1 (6.25%)	0.634	1 (9.10%)	0.95
G719X@ (%)	15 (93.75%)	0.579	10 (90.91%)	0.95
S768I (%)	5 (31.25%)	0.277	3 (27.27%)	0.984
# T790M Q-RT-PCR cut off value: 14.035; $ Exon 19 deletion Q-RT-PCR cut off value: 23.84; & L858R Q-RT-PCR cut off value: 19.015; @ G719X Q-RT-PCR cut off value: 16.09; * P<0.05

Table 5: Cell differentiation and EGFR mutation status

Differentiation	Dysplasia (n=4)	Well	Moderate	Poor	Undefined (n=8)	P value
		(n=2)	(n=13)	(n=5)
T790M#(%)	1 (25%)	1 (50%)	9 (69.23%)	3 (60%)	1 (12.5%)	0.079
Exon19 deletion$ (%)	4 (100%)	1 (50%)	10(76.92%)	5 (100%)	6 (75%)	0.729
L858R& (%)	4 (100%)	1 (50%)	6 (46.15%)	3 (60%)	7 (87.5%)	0.29
L861Q (%)	1 (25%)	0	1 (7.69%)	0	0	0.736
G719X@ (%)	4 (100%)	2 (100%)	12(92.31%)	5 (100%)	8 (100%)	0.882
S768I (%)	0	1 (50%)	2 (15.38%)	1 (20%)	3 (37.5%)	0.537
# T790M Q-RT-PCR cut off value: 14.035; $ Exon 19 deletion Q-RT-PCR cut off value: 23.84; & L858R Q-RT-PCR cut off value: 19.015; @ G719X Q-RT-PCR cut off value: 16.09; * P<0.05

Figure 1: Overall survival (OS) and disease-free survival (DFS) within EGFR mutation status in HCC patients. The cut-off value of T790M, Exon 19 deletion, L858R, and G719X are 14.035, 23.84, 19.015, and 16.09, separately.

DISCUSSION

60-85% of the tumor tissue in HCC showed overexpression of EGFR [7,16,17]. The proliferating activity, stage, intrahepatic dissemination, extrahepatic metastasis, and recurrence were all linked with EGFR expression [7,16]. The activation level of EGFR was reported with the relationship of susceptibility to levatinib17,18 use and was related to treatment response of tyrosine kinase inhibitor (TKI), gefitinib, and erlotinib [11-13,15]. Su, et al. ever reported that absence of EGFR exon 18-21 mutation in 89 HCC patients19. Lee, et al. also showed the same thing in 100 HCC patients [20]. However, they used the direct sequence to detect mutations, while the sequence is less sensitive than quantitative polymerase chain reaction (q-PCR) [21]. Bekaii-Saab, et al reported that 11% of HCC harbored a novel ERBB2 H878Y mutation in the activating domain of exon 21 [22]. The thirteen different missense mutations, including p.L730P, p.V742I, p.K757E, p.I780T, p.N808S, p.R831C, p.V851A, p.V897A, p.S912P, p.P937L, p.T940A, p.M947V, and p.M947T, and one single nucleotide polymorphism, p.Q787Q (CAG>CAA), in exons 19-23 were also reported in HCC samples [14].

In our study, we used ARMS and Scorpions real-time PCR, which would increase the sensitivity and specificity rate of detection. No one in our patient’s cohort received any TKI, including Sorafenib and Lenvatinib. We noticed that higher expression of T790M mutation, which had lower response rate in NSCLC treated with TKI, showed a good prognostic factor in overall survival, with statistics significant (P=0.001), in HCC patients receiving liver segmentectomy. EGFR T790M point mutation in lung cancer predicts poor outcomes due to EGFR-TKI resistance [23]. In an animal model, mice that express the T790M mutation alone develop tumors with a longer latency than mice that express both the T790M and L858R mutations, indicating that the T790M mutation is an oncogenic mutation that gives cancer cells a growth advantage in addition to being a cause of resistance to gefitinib/erlotinib [24]. In our HCC patient’s cohort, EGFR T790M mutation higher expression provided longer OS and a trend of longer disease-free survival (DFS), with a median DFS of 1193 days in the mutation group, and 291 days in the non-mutation group. It did not show statistically significant in DFS, which may be because of small case numbers.

HCC patients with higher expression of G719X mutation also had better OS (not reach vs 1308 days; P=0.036), and a trend of better DFS (4499 days vs 798 days; P=0.113) in our patients’ population. The phosphate-binding "P-loop" in the N-lobe, which arches over the triphosphate moiety to help coordinate ATP, contains G719X. G719X includes the substitutions G719S, G719A, G719C, and G719D. The P-loop becomes less flexible and the hydrophobic contacts that keep the C-helix in the inactive conformation are weakened when glycine is substituted for serine at position 719. This results in a 10-fold increase in kinase activity in comparison to WT EGFR [25,26]. G719X mutation confers to less sensitivity of the first generation TKI in lung cancer [26], however, there was no evidence that EGFR G719X mutation is related to oncogenesis. In our limited HCC patients’ cohort, higher expression of EGFR G719X mutation tended to show a better outcome, including DFS and OS, although 90% of patients carrying on G719X mutation had recurrent in our follow-up period.

EGFR overexpression is an early phenomenon in the pathogenesis of lung adenocarcinoma, while EGFR mutation (exon 19 deletion and L858R mutation) provides an increase in gene copy number and is related to tumor progression. EGFR exon 19 deletion and L858R mutation show better outcomes and are more sensitive to TKI in lung cancer [27]. Our HCC patients with higher expression of exon 19 deletions and L858R mutation had a trend of longer DFS after receiving hepatic segmentectomy. This phenomenon is opposite to the phenomenon in lung cancer. However, HCC patients with EGFR S768I had a trend of early progression and shorter DFS.

In a TP53-independent way, variations in EGFR were linked to the clinical outcome of HBV-related HCC and played a significant role in the control of p21 [28]. In the patient’s cohort, more than 85% of HCC patients with chronic hepatitis B showed T790M mutations and only one-fourth of patients with chronic hepatitis C had this mutation. Moreover, we found a negative relationship between T790M mutation and HCV carrier with statistic significant. That may relate to the pathogenesis of viral-induced cancer.

The limitation of our study is the small number of patients. Because our study is a retrospective analysis, the clinical data including α-fetoprotein were not complete. However, the survival difference in our study is valuable because of the very long-term follow-up.

CONCLUSION

Not only predict TKI response, but EGFR mutations also showed a predictive role in prognosis in HCC patients who did not receive TKI treatment. HCC patients with EGFR T790M or G719X mutations had better OS. Patients with exon 19 deletions, L858R, and G719X mutations showed trends of longer Disease Free Survival (DFS).

FUNDING

This research received no external funding.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

References:

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.
2. Petrick JL, Florio AA, Znaor A, et al. International trends in hepatocellular carcinoma incidence, 1978 -2012. Int J Cancer. 2020;147(2):317-330.
3. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71 (3):209-249.
4. Baecker A, Liu X, La Vecchia C, Zhang ZF. Worldwide incidence of hepatocellular carcinoma casese attributable to major risk factors. Eur J Cancer Prev. 2018; 27(3):205-212.
5. Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Review. Oncogene. 2010;29(36):4989-5005.
6. Daveau M, Scotte M, Francois A, et al. Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma. Mol Carcinog. 2003;36(3):130-141.
7. Zhao YN, Cao J, Wu FX, et al. Expression and significance of EGF mRNA and EGFR mRNA in hepatocellular carcinoma. Ai Zheng. 2004;23(7):762-766.
8. Ito Y, Takeda T, Sakon M, et al. Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma. Br J Cancer. 2001;84(10):1377-1383.
9. Sigismund S, Avanzato D, Lanzetti L. Emerging functions of the EGFR in cancer. Mol Oncol. 2018;12(1):3-20
10. Kira S, Nakanishi T, Suemori S, Kitamoto M, Watanabe Y, Kajiyama G. Expression of transforming growth factor alpha and epidermal growth factor receptor in human hepatocellular carcinoma. Liver. 1997;17(4):177-182.
11. DeCicco LA, Kong J, Rnger DP. Carcinogen-indeced alteration in liver epidermal growth factor receptor distribution during the promotion stage of hepatocarcinogenesis in rat. Cancer Lett. 1997;111(1-2):149-156.
12. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomized, phase 3 study. Lancet Oncol. 2011;12(8):735-42.
13. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomized phase 3 trial. Lancet Oncol. 2012;13(3):239-246.
14. Panvichian R, Tantiwetrueangdet A, Sornmayura P, Leelaudomlipi S. Missense Mutations in Exons 18-24 of EGFR in Hepatocellular Carcinoma Tissues. Biomed Res Int. 2015;2015:171845.
15. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, as inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. J Am Med. Assoc. 2003;290(16):2149-2158.
16. Ito Y, Takeda T, Sakon M, et al. Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma. Br J Cancer. 2004;84(10):1377-1383.
17. Altimari A, Fiorentino M, Gabusi E, et al. Investigation of ErbB1 and ErbB2 expression for therapeutic targeting in primary liver tumours. Dig Liver Dis. 2003;35(3):332-338.
18. Jin H, Shi Y, Lv Y, et al. EGFR activation limits the response of liver cancer to lenvatinib. Nature. 2021;595(7869):730-734.
19. Su MC, Lien, HC, Jeng YM. Absence of epidermal growth factor receptor exon 18-21 mutation in hepatocellular carcinoma. Cancer Lett. 2005; 224(1):117-121.
20. Lee SC, Lim SG, Soo R, et al. Lack of somatic mutations in EGFR tyrosine kinase domain in hepatocellular and nasopharyngeal carcinoma. Pharmacogenet Genomics. 2006;16(1):73-74.
21. Goto K, Satouchi M, Ishii G, et al. An evaluation study of EGFR mutation test utilized for non-small-cell lung cancer in the diagnostic setting. Ann Oncol. 2012;23(11):2914-2919.
22. Bekaii-Saab T, Williams N, Plass C, Calero MV, Eng C. A novel mutation in the tyrosine kinase domain of ERBB2 in hepatocellular carcinoma. BMC Cancer. 2006;6(1):1-5.
23. Bartholomew C, Eastlake L, Dunn P, Yiannakis D. EGFR targeted therapy in lung cancer; an evolving story. Respir Med Case Rep. 2017;20:137-140.
24. Regales L, Balak MN, Gong Y, et al. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors. PLoS ONE. 2007;2(8):e810.
25. Yun CH, Boggon TJ, Li Y, et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell. 2007;11(3):217-227.
26. Harrison PT, Vyse S, Huang PH. Rare epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer. Semin Cancer Biol. 2020;61:167-179.
27. Tang X, Varella-Garcia M, Xavier AC, et al. EGFR Abnormalities in the Pathogenesis and Progression of Lung Adenocarcinomas. Cancer Prev Res (Phila). 2008;1(3):192-200.
28. Han C, Liao X, Qin W, et al. EGFR and SYNE2 are associated with p21 expression and SYNE2 variants predict post-operative clinical outcomes in HBV-related hepatocellular carcinoma. Sci Rep. 2016;6:31237.