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.
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