Next-generation sequencing-based identification of EGFR and NOTCH2 complementary mutations in non-small cell lung cancer

Although targeted therapy has emerged as an effective treatment strategy for non-small cell lung cancer (NSCLC), some patients cannot benefit from such therapy due to the limited number of therapeutic targets. The present study aimed to identify mutated genes associated with clinicopathological characteristics and prognosis and to screen for mutations that are not concurrent with applicable drug target sites in patients with NSCLC. Tumor tissue and blood samples were obtained from 97 patients with NSCLC. A lung cancer-specific panel of 55 genes was established and analyzed using next-generation sequencing (NGS). The results obtained from the clinical cohort were compared with the NSCLC dataset from The Cancer Genome Atlas (TCGA). Subsequently, 25 driver genes were identified by taking the intersection of the 55 lung-cancer-specific genes with three databases, namely, the Catalog of Somatic Mutations in Cancer database, the Network of Cancer Genes database and Vogelstein''s list. Functional annotation and protein-protein interaction analysis were conducted on these 25 driver genes. The χ² test and logistic regression were used to evaluate the association between mutations in the 25 driver genes and the clinicopathological characteristics of 97 patients, and phosphatase and tensin homolog (PTEN) and kirsten rat sarcoma viral oncogene homolog (KRAS) were associated with stage at diagnosis and sex, respectively, while epidermal growth factor receptor (EGFR) was associated with sex, stage at diagnosis, metastasis, CEA and CYFRA21-1. Moreover, the association between the 25 driver gene mutations and overall survival were examined using Cox regression analysis. Age and Notch homolog 2 (NOTCH2) mutations were independent prognostic factors in TCGA dataset. The correlations between statistically significant mutations in EGFR, KRAS, PTEN and NOTCH2 were further examined, both in the clinical data and TCGA dataset. There was a negative correlation between EGFR and NOTCH2 mutations (correlation coefficient, −0.078; P=0.027). Thus, the present study highlights the importance of NOTCH2 mutations and might provide novel therapeutic options for patients with NSCLC who do not harbor EGFR mutations.     

非小细胞肺癌中EGFR和K-ras基因突变检测

目的:探讨不同类型非小细胞肺癌的EGFR和K-ras基因突变情况及其与肺癌相关临床病理特征的关系。方法:用厦门艾德ADxARMS试剂盒进行98例非小细胞肺癌患者肿瘤组织中EGFR（18,19,20,21外显子）基因和K-ras（12,13,61密码子）基因突变的检测。所有患者均未接受过吉非替尼的治疗。结果:98例样本中31例发生了EGFR基因突变,突变率为31.6%（31/98）,其中15例为19外显子缺失,13例为21 L858R外显子点突变,3例为20外显子突变,1例为18外显子突变。其中1例既有19外显子缺失突变,又有20外显子突变。腺癌中EGFR基因突变率较鳞癌、腺鳞癌、大细胞癌高。女性患者EGFR基因突变率较男性高。不吸烟患者EGFR基因突变率较吸烟患者高。低分化腺癌患者EGFR基因突变率较中、高分化患者高。21例发生了K-ras基因突变（21.4%）,其中12、13、61密码子均发现突变。突变率腺癌较鳞癌、腺鳞癌、大细胞癌高,与是否吸烟、患者性别、分化程度均无相关性。结论:非小细胞肺癌患者EGFR基因突变检出率较高,K-ras基因突变率较低,且两者不存在同时突变,EGFR基因突变与肺癌组织学类型、分化程度、性别等相关。K-ras基因突变与组织学类型相关。     

非小细胞肺癌患者血清EGFR基因突变循环DNA检测的临床研究

目的:探讨非小细胞肺癌患者血清EGFR基因突变循环DNA检测的临床意义。方法:选取2011年1月-2014年5月于我院接受治疗的124例非小细胞肺癌患者为研究对象,收集血清及组织标本DNA,检测DNA EGFR基因突变情况。结果:124例非小细胞肺癌患者血清EGFR基因检测结果显示:19号外显子突变11例,21号外显子突变8例,血清总检出19例,检出率为15.3%。组织样本中EGFR基因检测结果显示:19号外显子突变27例,21号外显子突变13例,血清总检出40例,检出率为32.3%。在组织样本中,79例男性EGFR基因突变检出14例,比例为17.7%;45例女性EGFR基因突变检出26例,比例为57.8%,相比具有显著性差异(P<0.05)。76例吸烟史患者检出突变16例,比例为21.1%;48例无吸烟史者检出突变24例,比例为50.0%,两者相比差异显著(P<0.05)。血清样本检测中结果与组织样本基本吻合,在性别和吸烟史方面患者基因突变有显著性差异(P<0.05),在癌症分期及类型方面无显著性差异(P>0.05)。结论:非小细胞肺癌患者血清循环DNA检测EGFR基因突变与肿瘤组织检测具有高度的一致性,这对肺癌患者的诊断、筛查和治疗有着重要的意义,为临床检测提供方便、有效的诊断方法。     

Survival and prognosis analyses of concurrent PIK3CA mutations in EGFR mutant non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors

In non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation, the prognostic impact of a concurrent Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) mutation was still unknown. Some studies have shown that EGFR mutant NSCLC patients treated with EGFR tyrosine kinase inhibitors (TKIs) when concurrent PIK3CA mutation have a worse prognosis and shorter survival time. This study conducted a retrospective analysis of NSCLC patients with EGFR mutant or concurrent PIK3CA mutations from January 2015 to October 2019 in the First Affiliated Hospital of Nanchang University. Relative to EGFR alone mutations (Single-Mt), we found that NSCLC patients with EGFR mutations coexisting with PIK3CA mutations (Double-Mt) treated with EGFR-TKIs had a shorter median time to progression (TTP): 7.8 months versus 10.9 months (Double-Mt versus Single-Mt, P = 0.001), and decrease in median overall survival (OS): 20.6 months versus 32.4 months (P < 0.001). The objective response rate (ORR) between Double-Mt and Single-Mt was 36.7% versus 61.9% (P = 0.044), disease control rates (DCR) was 80.1% versus 91.7% (P = 0.179). Obviously, EGFR-TKIs for EGFR mutate NSCLC patients when concurrent PIK3CA mutations have a worse prognosis and shorter survival time.     

Comprehensive investigation of oncogenic driver mutations in Chinese non-small cell lung cancer patients

Purpose

To determine the frequency of driver mutations in Chinese non-small cell lung cancer (NSCLC) patients.

Methods

Comprehensive mutational analysis was performed in 1356 lung adenocarcinoma, 503 squamous cell carcinoma, 57 adenosquamous lung carcinoma, 19 large cell carcinoma and 8 sarcomatoid carcinoma. The effect of EGFR tyrosine kinase inhibitors (TKIs) on EGFR-mutated lung adenocarcinoma patients after disease recurrence was investigated.

Results

Mutations in EGFR kinase domain, HER2 kinase domain, KRAS, BRAF, ALK, ROS1 and RET were mutually exclusive. In lung adenocarcinoma cases “pan-negative” for the seven above-mentioned driver mutations, we also detected two oncogenic EGFR extracellular domain mutations (A289D and R324L), two HER2 extracellular and transmembrane domain mutations (S310Y and V659E), one ARAF S214C mutation and two CD74-NRG1 fusions. Six (1.2%) FGFR3 activating mutations were identified in lung squamous cell carcinoma (five S249C and one R248C). There were three (15.8%) EGFR mutations and four (21.1%) KRAS mutations in large cell carcinoma. Three (37.5%) KRAS mutations were detected in sarcomatoid carcinoma. In EGFR-mutated lung adenocarcinoma patients who experienced disease recurrence, treatment with EGFR TKIs was an independent predictor of better overall survival (HR = 0.299, 95% CI: 0.172–0.519, P < 0.001).

Conclusion

We determined the frequency of driver mutations in a large series of Chinese NSCLC patients. EGFR TKIs might improve the survival outcomes of EGFR-mutated lung adenocarcinoma patients who experienced disease recurrence.     

Beclin1在非小细胞肺癌的表达及其与EGFR突变的关系

目的:观察Beclin1在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达情况,探讨其与EGFR突变的相关性.方法:分别采用DNA直接测序法、免疫组化法检测42例NSCLC患者EGFR突变情况及Beclin1表达情况,分析二者之间关系及临床意义.结果:与癌旁组织比较,NSCLC组织中Beclin1高表达(71.4％ vs 30.9％,P＜0.01),Beclin1的表达与吸烟史、病理类型有关(P =0.005,P=0.025).EGFR突变14例,其中高表达Beclin1 11例,低表达Beclin1 2例,突变率为57.9％(11/19) vs 16.7％(2/12),Beclin1的表达与EGFR突变具有显著正相关性(r=0.416,P=0.009).结论:NSCLC患者EGFR突变与Beclin1的表达具有正相关性,Beclin1表达情况在一定程度上有助于判断NSCLC的EGFR突变状况,可能为判断EGFR突变提供有意义的线索.     

EGFR突变型转移性非小细胞肺癌的靶向治疗疗效及安全性

目的 探讨表皮生长因子受体(EGFR)突变型转移性非小细胞肺癌的靶向治疗疗效及安全性.方法选取80例EGFR突变型转移性非小细胞肺癌患者为研究对象,根据治疗方法的不同将患者分为观察组和对照组,每组各40例.对照组采用放射治疗,观察组采用表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼联合放疗治疗,随访5～25个月,比较两组患者治疗4周后的疗效、治疗过程中的不良反应及随访结束时的生存情况.结果治疗4周后,观察组患者的治疗有效率和疾病控制率高于对照组(57.5％vs 35.0％,82.5％vs 62.5％),差异均有统计学意义(P﹤0.05).两组患者的不良反应均以1～2级为主,观察组患者的不良反应发生率为90.0％(36/40),与对照组的85.0％(34/40)比较,差异无统计学意义(P﹥0.05).观察组与对照组患者的中位生存时间分别为15.2个月(95％CI:12.357～18.697)和12.1个月(95％CI:9.354～15.652),差异有统计学意义(χ2=5.026,P=0.025).结论对于EGFR突变型转移性非小细胞肺癌,EGFR-TKI靶向治疗可以提高疗效,延长患者的生存时间.     

Frequent EGFR mutations in brain metastases of lung adenocarcinoma

Lung adenocarcinomas often metastasize to the brain, and the prognosis of patients with brain metastases is still very poor. The epidermal growth factor receptor (EGFR) gene is mutated in a considerable fraction of primary lung adenocarcinomas, in particular those with drastic response to EGFR tyrosine kinase inhibitors. The present study was designed to elucidate the prevalence of EGFR mutations in brain metastases and the timing of their occurrence during cancer progression. EGFR mutations were detected in 12 of 19 metastatic lung adenocarcinomas to the brain (63%). This frequency was higher than those in previous studies for EGFR mutations at various stages of lung adenocarcinoma in East Asia, including Japan (i.e., 20-55%). In 6 cases with EGFR mutations, the corresponding primary lung tumors were also examined for the mutations, and in all of them, the same types of EGFR mutations were detected also in the primary tumors. In 2 of them, second metastatic brain tumors in addition to the first ones were also available for analysis, and the same types of EGFR mutations were detected in both the first and second ones in both cases. These results indicate that EGFR mutations are present frequently in brain metastases and occur preceding brain metastasis. These findings will be highly informative for treatment of metastatic lung adenocarcinoma to the brain.     

超声引导粗针活检在非小细胞肺癌诊断及EGFR基因突变检测中的应用

目的:探讨超声引导下粗针活检在周围型非小细胞肺癌的诊断及EGFR基因突变检测中应用价值.方法:31例周围型非小细胞肺癌患者行超声引导下肺肿块粗针活检术,活检小标本行病理学检测及PCR法EGFR基因突变分析.结果:31例非小细胞肺癌活检小标本取材满意率100％,穿刺定性诊断符合率100％,EGFR基因检出率100％,并发症发生率3.2％.病理结果:腺癌25例、鳞癌2例、腺鳞癌3例、肉瘤样癌1例.31例非小细胞肺癌活检小标本中,检测到EGFR基因突变11例,阳性率35.5％.其中5例为第19外显子框内多核苷酸缺失,5例为第21外显子L858R突变,1例为联合突变L861Q/G719X(第18外显子G719X突变和第21外显子L861Q突变).结论:超声引导下肺肿块粗针活检术简便、安全、有效,能明确周围型非小细胞肺癌的诊断,是非小细胞肺癌获得肿瘤组织检测EGFR基因突变的可靠方法.     

非小细胞肺癌胸腔积液与配对肿瘤组织中EGFR基因突变检测的比较

目的:探讨非小细胞肺癌胸腔积液与配对肿瘤组织标本中EGFR基因突变检测结果的一致性,评价胸腔积液标本检测EGFR基因突变的应用价值.方法:收集非小细胞肺癌患者胸腔积液与配对肿瘤组织样本72例,采用ARMS方法,检测样本中EGFR基因第18~21外显子突变情况.结果:细胞学样本和组织学样本中EGFR基因突变阳性率分别为48.61%和51.39%,两者差异无统计学意义(P>0.05),二者一致率为92.11%,不一致率为7.89%.结论:二者的一致率较高,恶性胸水可以作为无法获得肿瘤组织的晚期非小细胞肺癌EGFR基因检测的有效样本.     

Analysis of EGFR Mutation Status in Algerian Patients with Non-Small Cell Lung Cancer

Background and objective: Epidermal growth factor receptor (EGFR) mutation status is used as a predictive biomarker for the tyrosine kinase inhibitors therapy in non-small cell lung cancer (NSCLC). The incidence of EGFR mutations appears to vary according to ethnic and geographical backgrounds. This retrospective study aimed to investigate the EGFR mutation status in Algerian NSCLC patients and its association with clinicopathological features. Methods: We examined the presence of EGFR mutations (Exons 19-21) in 58 unselected  NSCLC samples using PCR followed by direct sequencing. Results: The present study included 53 (91.4%) men and 5 (8.6%) women, with a median age of 59 (ranging from 44 to 94 years old). EGFR mutations were detected in 23 patients, with an overall rate of 39.6%. There were 21 (91.3%) cases with the exon-21 L585R single mutation and two (8.7%) with dual mutations of exon-19 deletions and L585R. EGFR mutations were more frequently found in patients with confirmed adenocarcinoma (14/27, 51.8%) than in non-adenomatous NCSCL subtypes (3/14, 21.4%; p=0.03). Furthermore, early stages of the disease were significantly associated with a higher rate of EGFR mutations (14/27, 51.8%) compared with those at  advanced stage (5/21, 23.8%; p=0.02). There were no significant differences in EGFR mutation frequency by age, gender, or smoking status. Conclusion: We found that Algerian NSCLC patients exhibited a high rate of EGFR mutations, which was quite similar to that in Asians population rather than Caucasian patients. Thus, TKI-based treatments may be more beneficial for Algerian patients with NSCLC. Further studies using a large number of patients are required to confirm our preliminary findings.     

Screening for EGFR mutations in lung cancer, a report from India

Epidermal growth factor receptor (EGFR) is one of the targeted molecular markers in many cancers including the lung malignancy. Genetic modifications such as deletions, insertions and Single Nucleotide Polymorphisms in the tyrosine kinase (TK) domain of EGFR is a common feature observed in most lung cancers. Gefitinib and erlotinib are commonly available therapeutic drugs which act as specific inhibitors for the tyrosine kinase domain of EGFR and associated with EGFR mutations in exons 18-21. However the prevalence of mutation varies among ethnicity, grade, age and gender. This is the first report on the prevalence of EGFR mutation in non-small cell lung cancer patients using DNA obtained from samples such as biopsy/cytology/pleural fluid and Fine Needle Aspiration (FNA), across India. We have screened for 29 somatic mutations which span exons 18, 19, 20 and 21 of EGFR gene using Scorpion probe based ARMS-PCR technique. DNA from 220 NSCLC tissue samples were analyzed for EGFR mutations and mutations were detected in 51.8% of the study population. Among the mutant positive cases, the deletions in exon 19 (52%) and a missense mutation L858R in exon 21 (26%) were most predominant. There was a significant increase in overall mutations (p = 0.01) as a function of age, mutation in exons 19 and 21 together (p = 0.003), mutations in exons 18, 19 and 21 (p = 0.04) and mutations in exons 18 and 19 (p = 0.03) in females. Mutations did not seem to significantly correlate metastases or disease progression. Mutations in exons [19] and 21 together were significant in non-smokers compared to smokers (p = 0.01) using Mann-Whitney tests. The study suggests high prevalence of EGFR positivity in NSCLC in Indian sub-population and provides opportunities for targeted therapies for this group.     

Identification of EGFR mutations in esophageal cancer

Background

It is well known that the prognosis for esophageal cancer is worse than for other digestive cancers in spite of multimodality treatment, and there is an urgent need to improve this situation. The epidermal growth factor receptor (EGFR) inhibitor, gefitinib, was approved in Japan to treat advanced non-small cell lung cancer patients and several papers have since reported that the successfully treated patients had genetic mutations in EGFR.

Purpose

The aim of this study was to investigate the existence of EGFR mutations in esophageal cancer cell lines and primary lesions, and also to explore the possibility of treating esophageal cancer using gefitinib.

Materials and Methods

Nineteen esophageal cancer cell lines were cultured and DNA was extracted using an ultracentrifugation method. Fifty cases of primary cancer and corresponding normal tissue samples were obtained and DNA was extracted using the same protocol. Nested PCR and DNA sequencing targeting exons 18, 19, 20 and 21 of EGFR were performed to investigate the presence of mutations in esophageal cancer cell lines and primary tumors.

Results

Three of the 19 cell lines had the same silent mutation at nucleotide 2607, a G-to-A substitution in exon 20. One of the 50 patients had an EGFR mutation in codon 719, resulting in an amino acid substitution from glycine to aspartic acid.

Conclusion

EGFR mutations in esophageal carcinoma are rare but do exist, and thus gefitinib could be included in esophageal cancer treatment regimens by selecting those patients who possess such mutations.     

非小细胞肺癌患者EGFR基因突变与吉非替尼临床疗效关联

目的:评估吉非替尼（Gefitinib）疗效和晚期难治性非小细胞肺癌（NSCLC）患者表皮生长因子受体（EGFR）突变情况的关联。方法:121名晚期难治性非小细胞肺癌患者,均是经过一线化疗方案失败的中晚期非小细胞肺癌患者,每日予以口服吉非替尼250mg治疗,直到疾病进展或出现不可耐受的毒副反应为止。在开始治疗之前检测EGFR18、19、21位点,于治疗期间进行常规检查和规范的随访。分析疗效、突变以及中位生存期之间的关联。结果:在115名有效随访的患者中,13例完全缓解,25例部分缓解,38例稳定,39例病情进展。疾病控制率66.3%。1年和2年生存率分别为59.7%和26.9%。中位生存期16个月。115人中有38例病人存在EGFR突变。EGFR突变的病人表现出对吉非替尼较敏感。不吸烟的女性患者有较好的疗效和较长的生存期。而患者年龄、一线化疗周期、肿瘤分期的组间差异对于生存期的影响都无显著差异。结论:吉非替尼在女性不吸烟非小细胞肺癌患者中疗效是确定的。西部女性肺腺癌患者拥有较高的突变率和较长的生存时间。     

阿帕替尼治疗EGFR野生型晚期非小细胞肺癌的疗效及安全性

目的:探索阿帕替尼治疗表皮生长因子受体(epidermal growthfactor receptor,EGFR)野生型晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的疗效及安全性.方法:回顾性纳入62例本中心符合纳入标准的、接受阿帕替尼单药治疗晚期NSCLC患者,分析其疗效及不良...     

Frequent EGFR mutations in noninvasive bronchioloalveolar carcinoma

Mutations of the epidermal growth factor receptor gene (EGFR) have been reported to be present in a considerable fraction of lung adenocarcinomas showing dramatic response to EGFR tyrosine kinase inhibitors. To clarify pathogenic significance of the mutations for the development of lung adenocarcinoma, we investigated stage I lung adenocarcinomas for the mutations. First, 107 cases of macrodissected stage I adenocarcinomas were examined for mutations in exons 18-21 of the EGFR gene. EGFR mutations were detected in 36 of the 107 cases (34%). In particular, among the stage I cases, the mutations were detected in 17 of 42 small-sized adenocarcinomas (相似文献   

FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib,a pan-EGFR family kinase inhibitor

Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [1] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [2] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [3] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [4] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [5] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.     

非小细胞肺癌患者细胞学标本EGFR基因突变检测及其临床病理意义

目的: 探讨细胞学标本在非小细胞肺癌(NSCLC)的诊断及个体化治疗中的临床应用价值。方法: 收集352例新鲜细胞学标本制片后,行常规HE染色;同时选择TTF-1、NapsinA、CK7、CEA、CD56、Syn、P63、CK5/6、WT-1、E-cadherin等抗体对来源不明的肿瘤细胞进行免疫细胞化学标记,并对明确诊断为NSCLC的病例,采用突变扩增阻滞系统(ARMS)检测表皮生长因子受体(EGFR)基因突变情况。结果: 352例患者中,345例有癌细胞。经临床及免疫细胞化学证实345例恶性细胞中,NSCLC有335例,且NSCLC细胞学标本中有302例DNA提取成功,占90.15%(302/335)。EGFR 基因检测结果显示,EGFR 共突变123例,总突变率为40.73%(123/302)。其中,第18、19、20、21外显子的突变率分别为0.99%(3/302)、19.21%(58/302)、0.66%(2/302)和19.87% (60/302); EGFR 18、19、21外显子突变占EGFR 突变总数的98.37%(121/123)显著高于EGFR 20外显子突变(P<0.05)。302例患者中,女性患者EGFR 突变率为54.35% (75/138),明显高于男性患者29.27%(48/164)(P<0.05);非吸烟患者EGFR 的突变率为51.49% (104/202),显著高于吸烟者19%(19/100)(P<0.05)。276例腺癌中EGFR 突变率44.20%(122/276);非腺癌EGFR 突变率4.34%(1/23);腺癌EGFR 突变率明显高于其他类型(P<0.05)。结论: 利用新鲜细胞学标本,结合免疫细胞化学标记和ARMS分子病理技术有助于晚期非小细胞肺癌的诊断,并为表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)个体化治疗提供可靠依据。