Epidermal Growth Factor Receptor Gene Mutation Status Research Articles

Heterogeneity and Clinical Effect of Epidermal Growth Factor Receptor in Primary Lung and Brain Metastases of Nonsmall Cell Lung Cancer

IntroductionThis study aimed to analyze the heterogeneity in epidermal growth factor receptor (EGFR) gene mutation and its impact on clinical outcomes in primary tumor and corresponding brain metastasis (BM) in nonsmall cell lung cancer (NSCLC). Materials and methodsPrimary pulmonary tumors and paired BMs of 27 NSCLC patients were surgically removed. All brain lesions were histologically confirmed as metastatic NSCLC. EGFR gene mutation status was detected by using amplification refraction mutation system. McNemar test was performed to compare EGFR mutation status between lung primary tumors and metastatic brain tumors and Kappa test was performed to quantify the agreement between the two. ResultsOf the 27 patients, nine cases were found to have EGFR mutations in BMs and 10 had a positive EGFR mutation status in primary lung tumor tissue. The rate of consistency of the matched tumor was 24/27 (88.9%). Among the three cases presenting EGFR mutational heterogeneity, two patients harbored an EGFR mutation in the primary tumor but not in the BMs; meanwhile, the last patient demonstrated the opposite pattern. Compared to patients with consistent EGFR mutations, patients with inconsistent mutations showed better outcomes. Further analysis revealed that the two patients whose EGFR mutant-type primary tumor progressed to wild-type cerebral metastatic tumor had longer overall survival than the patient whose EGFR wild-type primary tumor progressed to mutant-type brain metastatic tumor. ConclusionsHeterogeneity of EGFR mutation status was observed between primary NSCLC and paired BM. Patients possessing a wild-type EGFR mutation in BM might have better outcomes, especially those with transition from mutant to wild-type.

Association between epidermal growth factor receptor gene mutation status and short-term efficacy of first-line platinum-containing chemotherapy in advanced non-small cell lung cancer.

It remains undetermined whether there is an explicit association between the epidermal growth factor receptor (EGFR) gene mutation status and chemotherapy efficacy in non-small cell lung cancer (NSCLC) patients with advanced stages. Thus, the aim of the present retrospective study was to investigate the possible association between EGFR gene mutation status and the efficacy of first-line chemotherapy in patients with advanced NSCLC. In total, 52 patients who were diagnosed with NSCLC at Changzhou Tumor Hospital (Changzhou, China) from January 2015 to December 2018 were enrolled. All 52 patients received pemetrexed combined with platinum chemotherapy, for 21 days per cycle. After two cycles of treatment, the short-term clinical efficacy was assessed according to the Response Evaluation Criteria in Solid Tumours 1.1 guidelines. The objective response rate (ORR), disease control rate (DCR) and progression-free survival (PFS) rate were calculated at the end of the study (December 31, 2019). These patients also underwent second-generation gene sequencing before the potential association between mutations in the EGFR gene and chemotherapy efficacy was analyzed. In this group of patients, 25 cases (48.1%) were found to be harboring EGFR gene mutation, whilst 27 cases (51.9%) expressed wild-type EGFR. After receiving the first-line chemotherapy regimen, the ORR was determined to be 36.5%, the DCR was 71.2%, whereas the PFS period was 207 days. Following first-line chemotherapy, the DCR of patients with EGFR mutations (52%) was higher compared with those in patients harboring the wild-type EGFR (22%). By contrast, the PFS (260 days) of patients with EGFR mutations was longer compared with those in patients harboring wild-type EGFR (100 days). These differences were statistically significant (P<0.05). Multivariate analysis revealed that EGFR gene mutation was an independent predictor of PFS in patients with advanced NSCLC (P<0.05). To conclude, data from the present study suggest that EGFR gene mutation has independent predictive value for the efficacy of first-line chemotherapy in patients with advanced NSCLC.

Detection of circulating tumor cells with chromosomes 7 and 8 polysomy in non-small cell lung cancer and its correlation with epidermal growth factor receptor mutations in cancer tissue

Objective: To investigate the value of chromosomes 7 and 8 polysomy in circulating tumor cells (CTCs) for the diagnosis of non-small cell lung cancer, and the correlation of CTCs with clinical pathological characteristics and epidermal growth factor receptor (EGFR) mutations in cancer tissue. Methods: Fifty-seven patients with non-small cell lung cancer and 21 patients with benign lung diseases were enrolled at Beijing Chaoyang Hospital, Capital Medical University, Beijing, China from November 2017 to October 2020. Negative enrichment combined with immunofluorescence in situ hybridization (imFISH) was used to identify CTCs polysomy on chromosomes 7 and 8. EGFR mutations in 56 lung cancer patients was detected using ARMS-PCR. Results: CTCs were detected in 93.0% (53/57) of non-small cell lung cancers and 28.6% (6/21) benign lung lesions. The difference between lung cancer patients and the control cohort was statistically significant (P<0.01). Receive operator curve (ROC) analyses showed that, when the cut-off value was 1 cell/3.2 mL, Youden index had the highest sensitivity of 93.0% and specificity of 71.4% (AUC=0.906, 95%CI:0.833-0.980, P<0.01). The positive rate of CTCs in stage Ⅲ-Ⅳ cancers was significantly higher than that in stage Ⅰ-Ⅱ (P=0.023). No significant correlation was observed between positive rate of CTCs or chromosome polysomy and age, gender, smoking status, pathologic types and EGFR mutation status. The number of CTCs in EGFR mutated group was higher than that in the non-mutated group (6.5±1.1 vs. 3.7±0.7, P=0.045). The detection rate for CTCs ≥5 in the EGFR mutated group was also higher than the EGFR non-mutated group (52.0% vs. 19.4%,P=0.010). Conclusion: Detection of CTCs with chromosomes 7 and 8 polysomy has potential value in auxiliary diagnosis of non-small cell lung cancer, and the number of CTCs is correlated to TNM stage and EGFR gene mutation status.

Development and validation of a multivariable predictive model for EGFR gene mutation status in patients with lung adenocarcinoma.

Detection of epidermal growth factor receptor (EGFR) is one real dilemma owing to the non-sufficient tissue for testing EGFR mutations in lung adenocarcinoma. A model for predicting EGFR mutations would be helpful for clinical decisions in those patients. A retrospective cohort of 1,196 patients diagnosed with lung adenocarcinoma was investigated between December 1, 2017, and December 31, 2019, in Renji Hospital, Shanghai, China. All patients were tested for EGFR mutations (amplification refractory mutation system, n=1,144; next-generation sequencing, n=52). Of 1,196 patients with lung adenocarcinoma, 944 met the inclusion criteria. A nomogram model was developed based on 567 patients and validated in 377 patients. Variables associated with EGFR mutations were age, sex, smoking history, lepidic predominant subtype, solid predominant subtype, mucinous adenocarcinoma, Ki67 expression, lobulation, solid texture in radiology, and pleural retraction. The nomogram based on the model performed well in the development group (c-index 0.789, 95% CI: 0.751-0.827), and the validation group (c-index 0.809, 95% CI: 0.771-0.847). At the probability cut-point of 0.7, the diagnostic efficiency was 82.7% in patients with NGS liquid biopsy. Decision curve analysis further confirmed the clinical usefulness of the nomogram, which showed that predicting the EGFR mutations probability applying this nomogram would be better than having all patients or none patients use this nomogram. A high probability group (>0.7) by nomogram model may suggest a high possibility of EGFR mutation, if tissue is limited, NGS-based ctDNA with liquid biopsy could be implemented effectively.

Hormone receptor expression correlates with EGFR gene mutation in lung cancer in patients with simultaneous primary breast cancer.

BackgroundThe coexistence of double primaries of lung cancer (LC) and breast cancer (BC) are not uncommon in women, but there has been limited research conducted of their molecular association. To decipher the internal pathogenesis of LC in patients with concurrent BC and LC, this study explored the clinical factors and relationship between hormone receptor (HR) expression and epidermal growth factor receptor (EGFR) gene mutation.MethodsThe clinicopathological characteristics of 400 female patients clinically diagnosed with double primary LC and BC at Fudan University Shanghai Cancer Center were collected. Pathological discrimination was performed to further confirm the double primaries in patients with available tissues. LC samples were then examined to detect EGFR gene mutation status by PCR-based assays and HR expression by immunohistochemistry (IHC). As a control cohort, the characteristics of 114 consecutive patients with LC only were compared with the double-primary patient group.ResultsA total of 169 patients were pathologically confirmed with simultaneous LC and BC between January 2010 and October 2018. The dominant LC subtype was adenocarcinoma (ADC) (95.1%), and invasive ductal carcinoma (IDC) was the main BC subtype (71.0%). Synchronous and metachronous double primary BC-LC cases accounted for 39.1% and 60.9% of the patients, respectively. The absence of family cancer history was associated with a shorter interval between the two primary cancer diagnoses. Among 64 patients with EGFR mutations, 34.4% had HR-positive LC tissue, compared with 0/24 (0%) of those with EGFR wild-type LC (P<0.001). All of the patients with positive HR expression harbored an activating EGFR mutation (n=22); however, no correlation was observed in the control cohort.ConclusionsDouble primary BC-LC patients have distinctive clinicopathological features compared to those with LC only. The expression of HRs is significantly correlated with EGFR mutation status of LC tissues.

EGFR mutation genotyping and ALK status determination in liquid-based cytology samples of non-small cell lung cancer.

Personalised medicine for primary lung cancers (PLCs) requires molecular analysis of cancer tissue or cells. The primary objective of the present prospective study was to assess the concordance between epidermal growth factor receptor (EGFR) gene mutation detection and echinoderm microtubule-associated protein-like (EML) 4-anaplastic lymphoma kinase protein (ALK) expression using liquid-based cytology (LBC) samples and matched histology samples of PLC patients. A total of 117 patients who underwent surgical resection of non-small cell PLC were enrolled. Cytological specimens scratched from the resected PLC lesion were fixed in CytoRich Red. DNA extracted from LBC samples was examined for EGFR gene mutations. Anaplastic lymphoma kinase arrangement was analysed by immunostaining and fluorescence in situ hybridisation. Our patient cohort comprised 93 cases of adenocarcinoma, 16 squamous cell carcinoma, three adenosquamous carcinoma, two large cell neuroendocrine carcinoma, one pleomorphic carcinoma and two other cases. Sixty-six (58.4%) LBC samples harboured EGFR gene mutations. The overall concordance rate in EGFR gene mutation status, including minor mutations, between histologic and paired LBC specimens (N = 105) was 100%. The overall concordance rate of EGFR gene mutation status, including minor mutations and ALK status according to immunostains between histologic and paired LBC specimens, was 100% (105/105) and 100% (48/48), respectively. Genotyping and protein expression studies can be reliably performed using LBC samples prepared with CytoRich Red. Analysis of such samples may guide individual therapy in PLC patients.

Application value of 18F-FDG PET/CT in predicting EGFR mutation status in non-small cell lung cancer

Lung cancer is a malignant tumor with the high morbidity and mortality in the world, and non-small cell lung cancer (NSCLC) is the most common type. The emergence of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) in recent years has provided new treatment option for NSCLC patients. The efficacy of EGFR-TKI is closely related to the EGFR mutation status, but the current detection methods for gene mutation have certain limitations. As a non-invasive method, 18F-fluorodeoxyglucose (FDG) PET/CT shows a certain potential in the detection of EGFR gene mutation status. In this paper, the recent research and progress of PET/CT in predicting the mutation status of EGFR gene are reviewed. Key words: Carcinoma, non-small-cell lung; Mutation; Receptor, epidermal growth factor; Positron-emission tomography; Tomography, X-ray computed; Deoxyglucose; Trends

Clinicopathological characteristics with EGFR, ALK, ROS1 genetic alternation and prognostic analysis of primary lymphoepithelioma-like carcinoma

Background: Primary lymphoepithelioma-like carcinoma (LELC) is a rare form of lung cancer, since genetic alternations participate in the carcinogenesis in lung cancer, whether critical driver genes such as ROS1 fusion, anaplastic lymphoma kinase (ALK) rearrangement and epidermal growth factor receptor ( EGFR ) mutation play roles in LELC remains unclear. Methods: We collected a total of 30 LELC samples for genetic and prognosis analysis retrospectively in Shanghai Chest Hospital, EGFR gene mutation, ALK rearrangement and ROS1 fusion status were extracted from digital database. Clinicopathological characteristics with genetic profiling and survival were analyzed. Results: All samples appeared negative for genes ( EGFR, ALK and ROS1 ) alternations detection. Female gender acted as an independently prognostic factor in poorer disease-free survival (DFS) (P=0.034), and tumors locate in the left lobe associate with worse DFS in univariate analysis (significant trend, P=0.051), moreover, serum positive NSE level also indicate a shorter DFS after adjustment (significant trend, P=0.057). Most of LELC are diagnosed at early stage, with 90.0% (27/30) patients obtained the opportunities for surgery. Conclusions: Since no classical genetic alternations appeared in present study, more investigations of other genes distributions should be explored in the future for better understanding of this rare subtype of lung cancer. Serum positive neuron-specific enolase (NSE) level seemed to be a prognostic biomarker for DFS in LELC patients.

Clinicopathological characteristics with EGFR, ALK, ROS1 genetic alternation and prognostic analysis of primary lymphoepithelioma-like carcinoma.

BackgroundPrimary lymphoepithelioma-like carcinoma (LELC) is a rare form of lung cancer, since genetic alternations participate in the carcinogenesis in lung cancer, whether critical driver genes such as ROS1 fusion, anaplastic lymphoma kinase (ALK) rearrangement and epidermal growth factor receptor (EGFR) mutation play roles in LELC remains unclear.MethodsWe collected a total of 30 LELC samples for genetic and prognosis analysis retrospectively in Shanghai Chest Hospital, EGFR gene mutation, ALK rearrangement and ROS1 fusion status were extracted from digital database. Clinicopathological characteristics with genetic profiling and survival were analyzed.ResultsAll samples appeared negative for genes (EGFR, ALK and ROS1) alternations detection. Female gender acted as an independently prognostic factor in poorer disease-free survival (DFS) (P=0.034), and tumors locate in the left lobe associate with worse DFS in univariate analysis (significant trend, P=0.051), moreover, serum positive NSE level also indicate a shorter DFS after adjustment (significant trend, P=0.057). Most of LELC are diagnosed at early stage, with 90.0% (27/30) patients obtained the opportunities for surgery.ConclusionsSince no classical genetic alternations appeared in present study, more investigations of other genes distributions should be explored in the future for better understanding of this rare subtype of lung cancer. Serum positive neuron-specific enolase (NSE) level seemed to be a prognostic biomarker for DFS in LELC patients.

Identifying epidermal growth factor receptor mutation status in patients with lung adenocarcinoma by three-dimensional convolutional neural networks.

Genetic phenotype plays a central role in making treatment decisions of lung adenocarcinoma, especially the tyrosine-kinase-inhibitors-sensitive mutations of the epidermal growth factor receptor (EGFR) gene. We constructed three-dimensional convolutional neural networks (CNN) to analyze underlying patterns in CT images that could indicate that EGFR gene mutation status but are invisible to human eyes. From 2012 to 2015, 503 Chinese patients with lung adenocarcinoma that had underwent surgery were included. Pathological types and EGFR mutation status were tested from surgical resections. EGFR mutations (exon 19 deletion or exon 21 L858R) were found in 215/345 (62.3%) and 91/158 (57.6%) patients in the training and independent validation set, respectively. CT images were taken before any invasive operation. The patients were randomly chosen to train the CNNs or validate the CNNs' performance. The performance was quantified using area under receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy. The CNNs showed an AUC of 0.776 (range: 0.702-0.849, p< 0.0001) in the independent validation set and a fusion model of CNNs and clinical features (sex and smoking history) showed an AUC of 0.838 (range: 0.778-0.899, p< 0.0001), accuracy of 77.2%, sensitivity of 75.8% and specificity of 79.1% at the best diagnostic decision point. The CNN exhibits potential ability to identify EGFR mutation status in patients with lung adenocarcinoma which might help make clinical decisions. The CNN showed some diagnostic power and its performance could be further improved by increasing the training set, optimizing the network structure and training strategy. Medical image based CNN has the potential to reflect spatial heterogeneity.

Erratum to factors associated with the prognosis and long-term survival of patients with metastatic lung adenocarcinoma: a retrospective analysis.

[This corrects the article DOI: 10.21037/jtd.2018.03.143.].

Factors associated with the prognosis and long-term survival of patients with metastatic lung adenocarcinoma: a retrospective analysis.

Outcome of patients with advanced non-small cell lung cancer (NSCLC) treated with platinum-based doublet chemotherapy is usually poor, with overall survival ranges from 8-13 months. However, the overall survival is improved to 21-28 months in the era of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). This study aimed to explore the prognostic factors to predict long-term survival in the era of EGFR TKI. A total 1,030 patients were studied who had been diagnosed with metastatic adenocarcinoma of lung from January 2005 to December 2009 at Linkou Chang Gung Memorial Hospital. Long-term survivors (LTS) were defined as patients who survived five years or more after the initial diagnosis of stage IV disease. Data on patient age, gender, smoking status, performance status at diagnosis, TNM stage, metastatic pattern, number of metastases, and organs with tumor involvement, EGFR gene mutation status, types of treatment received, EGFR TKI treatment and longest EGFR TKI duration were retrospectively obtained from medical charts. We calculated the odds ratio (OR) of long-term survival from collected clinical parameters to predict long-term survival. In this large retrospective study, we reported a five-year survival of 5.0% among patients with metastatic adenocarcinoma of lung. A total 52 LTS and 978 non-LTS were identified. Patients had more contralateral or pleural/pericardial metastases in the LTS group than in the non-LTS group (51.9% vs. 19.0%, P<0.001), while less extrathoracic spread than in the non-LTS group (42.3% vs. 79.6%, P<0.001). The mutation of the EGFR gene was more frequent in the LTS group than in the non-LTS group (19.2% vs. 7.1%, P=0.006). In conclusion, our results suggest that an age younger than 60 years, absence of extrathoracic spread and EGFR TKI treatment duration of more than one year play an important role in the long-term for survivors who survive for more than 5 years.

Comparison of EGFR mutation between supernatant and pellets of malignant pleural effusion from patients with advanced non-small cell lung cancer

Objective To compare the epidermal growth factor receptor (EGFR) mutation differences between supernatant and pellets of malignant pleural effusion (MPE) in advanced non-small cell lung cancer (NSCLC) patients and provide the evidence for clinical accurate detection. Methods A total of 386 consecutive MPE specimens were collected in the study (202 in experimental group and 184 in validation group). Specimens in experimental group were divided into 4 groups depending on tumors cell concentration in the samples: zero cell (n=77), (1-5)×104/ml cells (n=43), (6-10)×104/ml cells (n=52) and >10×104/ml cells group (n=30). Amplification refractory mutation system (ARMS) was performed to detect EGFR gene mutation in supernatant and pellets of each individual case respectively, and the EGFR mutation positive rates were compared between the two groups. EGFR mutation test was carried out using either supernatant or pellets in validation group to verify the established method. Results In the experimental group, the total EGFR mutation positive rate was 30.7% (62/202). In the zero cell group, EGFR mutation positive rate was higher in supernatant than that in pellets (37.6% vs. 33.8%). In the (1-5)×104/ml cells group and (6-10)×104/ml cells group, EGFR mutation positive rate was equal but not concordant between supernatant and pellets (25.6% and 21.1% respectively). In the >10×104/ml cells group, EGFR mutation positive rate was equal (33.3%) with 100% concordance between supernatant and pellets. In the validation group (n=184), EGFR mutation rate was 32.7% (36/110) in supernatant and 32.4% (24/74) in pellets, and there was no statistical significance (χ2=0.02, P=0.97). Conclusion Tumor cell free MPE specimens from patients with advanced NSCLC are suitable for EGFR mutation test. Heterogeneity of MPE results in difference with respect to EGFR gene mutation status between cell-free supernatant and pellet. Pathological evaluation based on the quantity and quality of tumor cells in MPE patients prior to test and optional samples selection are necessary to increase EGFR mutation positive rate. Key words: Pleural effusion, malignant; Receptor, epidermal growth factor; Mutation

Icotinib versus whole-brain irradiation in patients with EGFR-mutant non-small-cell lung cancer and multiple brain metastases (BRAIN): a multicentre, phase 3, open-label, parallel, randomised controlled trial

For patients with non-small-cell lung cancer (NSCLC) and multiple brain metastases, whole-brain irradiation (WBI) is a standard-of-care treatment, but its effects on neurocognition are complex and concerning. We compared the efficacy of an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), icotinib, versus WBI with or without chemotherapy in a phase 3 trial of patients with EGFR-mutant NSCLC and multiple brain metastases. We did a multicentre, open-label, parallel randomised controlled trial (BRAIN) at 17 hospitals in China. Eligible participants were patients with NSCLC with EGFR mutations, who were naive to treatment with EGFR-TKIs or radiotherapy, and had at least three metastatic brain lesions. We randomly assigned participants (1:1) to either icotinib 125 mg orally (three times per day) or WBI (30 Gy in ten fractions of 3 Gy) plus concurrent or sequential chemotherapy for 4-6 cycles, until unacceptable adverse events or intracranial disease progression occurred. The randomisation was done by the Chinese Thoracic Oncology Group with a web-based allocation system applying the Pocock and Simon minimisation method; groups were stratified by EGFR gene mutation status, treatment line (first line or second line), brain metastases only versus both intracranial and extracranial metastases, and presence or absence of symptoms of intracranial hypertension. Clinicians and patients were not masked to treatment assignment, but individuals involved in the data analysis did not participate in the treatments and were thus masked to allocation. Patients receiving icotinib who had intracranial progression only were switched to WBI plus either icotinib or chemotherapy until further progression; those receiving icotinib who had extracranial progression only were switched to icotinib plus chemotherapy. Patients receiving WBI who progressed were switched to icotinib until further progression. Icotinib could be continued beyond progression if a clinical benefit was observed by the investigators (eg, an improvement in cognition or intracranial pressure). The primary endpoint was intracranial progression-free survival (PFS), defined as the time from randomisation to either intracranial disease progression or death from any cause. We assessed efficacy and safety in the intention-to-treat population (all participants who received at least one dose of study treatment), hypothesising that intracranial PFS would be 40% longer (hazard ratio [HR] 0·60) with icotinib compared with WBI. This trial is registered with ClinicalTrials.gov, number NCT01724801. Between Dec 10, 2012, and June 30, 2015, we assigned 176 participants to treatment: 85 to icotinib and 91 to WBI. 18 withdrew from the WBI group before treatment, leaving 73 for assessment. Median follow-up was 16·5 months (IQR 11·5-21·5). Median intracranial PFS was 10·0 months (95% CI 5·6-14·4) with icotinib versus 4·8 months (2·4-7·2) with WBI (equating to a 44% risk reduction with icotinib for an event of intracranial disease progression or death; HR 0·56, 95% CI 0·36-0·90; p=0·014). Adverse events of grade 3 or worse were reported in seven (8%) of 85 patients in the icotinib group and 28 (38%) of 73 patients in the WBI group. Raised concentrations of alanine aminotransferase and rash were the most common adverse events of any grade in both groups, occurring in around 20-30% of each group. At the time of final analysis, 42 (49%) patients in the icotinib group and 37 (51%) in the WBI group had died. 78 of these patients died from disease progression, and one patient in the WBI group died from thrombogenesis related to chemotherapy. In patients with EGFR-mutant NSCLC and multiple brain metastases, icotinib was associated with significantly longer intracranial PFS than WBI plus chemotherapy, indicating that icotinib might be a better first-line therapeutic option for this patient population. Guangdong Provincial Key Laboratory of Lung Cancer Translational Medicine, National Health and Family Planning Commission of China, Guangzhou Science and Technology Bureau, Betta Pharmaceuticals, and the Chinese Thoracic Oncology Group.

The role of metabolic tumor volume (MTV) measured by [18F] FDG PET/CT in predicting EGFR gene mutation status in non-small cell lung cancer.

Many noninvasive methods have been explored to determine the mutation status of the epidermal growth factor receptor (EGFR) gene, which is important for individualized treatment of non-small cell lung cancer (NSCLC). We evaluated whether metabolic tumor volume (MTV), a parameter measured by [18F] fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) might help predict EGFR mutation status in NSCLC. Overall, 87 patients who underwent EGFR genotyping and pretreatment PET/CT between January 2013 and September 2016 were reviewed. Clinicopathologic characteristics and metabolic parameters including MTV were evaluated. Univariate and multivariate analyses were used to assess the independent variables that predict mutation status to create prediction models. Forty-one patients (41/87) were identified as having EGFR mutations. The multivariate analysis showed that patients with lower MTV (MTV≤11.0 cm3, p=0.001) who were non-smokers (p=0.037) and had a peripheral tumor location (p=0.033) were more likely to have EGFR mutations. Prediction models using these criteria for EGFR mutation yielded a high AUC (0.805, 95% CI 0.712–0.899), which suggests that the analysis had good discrimination. In conclusion, NSCLC patients with EGFR mutations showed significantly lower MTV than patients with wild-type EGFR. Prediction models based on MTV and clinicopathologic characteristics could provide more information for the identification of EGFR mutations.

Mutations of the Epidermal Growth Factor Receptor Gene in Triple-Negative Breast Cancer.

PurposeEpidermal growth factor receptor (EGFR) is considered a potential therapeutic target for anti-EGFR therapy in triple-negative breast cancer (TNBC). However, the frequency of EGFR gene mutation in TNBC is low and varies with ethnicity. This study aimed to investigate the incidence of EGFR gene mutation in TNBC.MethodsEGFR protein expression was evaluated by immunohistochemistry in tissue microarrays of 493 TNBC cases using four different primary antibodies, which included mutation-specific antibodies. For cases with an immunoreactivity level ≥1+, we performed pyrosequencing analysis for EGFR gene mutation. A case was considered mutation-positive when its mutation frequency minus its limit of detection (LOD) was >10%. Cases with mutation frequency higher than LOD were assessed for EGFR gene mutation status using the Cobas assay and by peptide nucleic acid-mediated polymerase chain reaction (PNA-clamping).ResultsAmong 493 TNBCs, 148 (30.0%) exhibited staining ≥1+ for EGFR, including 78 with 1+, 49 with 2+, and 21 with 3+. Positive EGFR expression (≥2+) was significantly associated with lymphovascular invasion (p=0.010), but not with overall survival (p=0.444) or disease-free survival (p=0.388). None of the 493 TNBCs harbored an EGFR gene mutation. Among 148 cases with an EGFR staining result ≥1+, five (3.4%) showed mutation frequencies (4.4%–10.9%) higher than LOD (2.6%–4.3%) in exons 19 (L747_P753>Q) or 21 (L858R and L861Q) as determined by pyrosequencing. However, Cobas and PNA-clamping failed to detect the presence of EGFR gene mutation in these five cases.ConclusionNo activating mutation of EGFR gene of clinical significance was observed in 148 TNBC cases using three commercially available methods. Thus, EGFR gene mutation appears to be an extremely rare event in patients with TNBC.

Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and identification of pitfalls using FFPE tissue.

In the growing field of personalised medicine, the analysis of numerous potential targets is becoming a challenge in terms of work load, tissue availability, as well as costs. The molecular analysis of non-small cell lung cancer (NSCLC) has shifted from the analysis of the epidermal growth factor receptor (EGFR) mutation status to the analysis of different gene regions, including resistance mutations or translocations. Massive parallel sequencing (MPS) allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small formalin-fixed, paraffin-embedded (FFPE) biopsies. In this study, we compared and evaluated currently used MPS platforms for their application in routine pathological diagnostics. We initiated a first round-robin testing of 30 cases diagnosed with NSCLC and a known EGFR gene mutation status. In this study, three pathology institutes from Germany received FFPE tumour sections that had been individually processed. Fragment libraries were prepared by targeted multiplex PCR using institution-specific gene panels. Sequencing was carried out using three MPS systems: MiSeq™, GS Junior and PGM Ion Torrent™. In two institutes, data analysis was performed with the platform-specific software and the Integrative Genomics Viewer. In one institute, data analysis was carried out using an in-house software system. Of 30 samples, 26 were analysed by all institutes. Concerning the EGFR mutation status, concordance was found in 26 out of 26 samples. The analysis of a few samples failed due to poor DNA quality in alternating institutes. We found 100% concordance when comparing the results of the EGFR mutation status. A total of 38 additional mutations were identified in the 26 samples. In two samples, minor variants were found which could not be confirmed by qPCR. Other characteristic variants were identified as fixation artefacts by reanalyzing the respective sample by Sanger sequencing. Overall, the results of this study demonstrated good concordance in the detection of mutations using different MPS platforms. The failure with samples can be traced back to different DNA extraction systems and DNA quality. Unknown or ambiguous variations (transitions) need verification with another method, such as qPCR or Sanger sequencing.

Classification of Epidermal Growth Factor Receptor Gene Mutation Status Using Serum Proteomic Profiling Predicts Tumor Response in Patients with Stage IIIB or IV Non-Small-Cell Lung Cancer.

Objectives Epidermal growth factor receptor (EGFR) gene mutations in tumors predict tumor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissue for mutation analysis is challenging. Here, we aimed to detect serum peptides/proteins associated with EGFR gene mutation status, and test whether a classification algorithm based on serum proteomic profiling could be developed to analyze EGFR gene mutation status to aid therapeutic decision-making.Patients and MethodsSerum collected from 223 stage IIIB or IV NSCLC patients with known EGFR gene mutation status in their tumors prior to therapy was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinProTools software. Differences in serum peptides/proteins between patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes were detected in a training group of 100 patients; based on this analysis, a serum proteomic classification algorithm was developed to classify EGFR gene mutation status and tested in an independent validation group of 123 patients. The correlation between EGFR gene mutation status, as identified with the serum proteomic classifier and response to EGFR-TKIs was analyzed.ResultsNine peptide/protein peaks were significantly different between NSCLC patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes in the training group. A genetic algorithm model consisting of five peptides/proteins (m/z 4092.4, 4585.05, 1365.1, 4643.49 and 4438.43) was developed from the training group to separate patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes. The classifier exhibited a sensitivity of 84.6% and a specificity of 77.5% in the validation group. In the 81 patients from the validation group treated with EGFR-TKIs, 28 (59.6%) of 47 patients whose matched samples were labeled as “mutant” by the classifier and 3 (8.8%) of 34 patients whose matched samples were labeled as “wild” achieved an objective response (p<0.0001). Patients whose matched samples were labeled as “mutant” by the classifier had a significantly longer progression-free survival (PFS) than patients whose matched samples were labeled as “wild” (p=0.001).ConclusionPeptides/proteins related to EGFR gene mutation status were found in the serum. Classification of EGFR gene mutation status using the serum proteomic classifier established in the present study in patients with stage IIIB or IV NSCLC is feasible and may predict tumor response to EGFR-TKIs.

Epidermal growth factor receptor gene mutation status and its association with clinical characteristics and tumor markers in non-small-cell lung cancer patients in Northwest China.

This study was conducted to investigate the mutation status of epidermal growth factor receptor (EGFR) and its association with clinical characteristics and tumor markers in non-small-cell lung cancer (NSCLC) patients from the Xinjiang Uygur Autonomous Region in China. We enrolled 51 cases of NSCLC patients who received radical surgical treatment in the First Affiliated Hospital of Xinjiang Medical University. Quantitative polymerase chain reaction was applied to detect exons 18, 19, 20 and 21 of the EGFR gene in tumor tissues. Multiple tumor markers, including carcinoembryonic antigen (CEA), were assessed preoperatively. The EGFR-positive rate was 49.02% (25/51), with a mutation rate of 8% (2/25) in exon 18, 52% (13/51) in exon 19, 40% (10/51) in exon 21 and no mutations in exon 20. The positive mutation rate in men and women was 37.5% (12/32) and 68.42%, respectively (13/19), with a statistically significantly higher rate in women (P<0.05). There were also statistically significant differences among adenocarcinoma, adenosquamous carcinoma and squamous cell carcinoma cases (P<0.05), while no statistically significant differences were observed in adenocarcinoma cases regarding degree of differentiation, lymph node metastasis and TNM stage (P>0.05). There was a statistically significant association between the EGFR gene mutation status and the preoperative serum CEA level (P<0.05). The mutation rate of the EGFR gene was 68.42% in female lung adenocarcinoma patients, which supports the application of targeted therapy in such cases. However, whether it is possible to obtain information regarding targeted therapy through measuring the level of serum CEA for NSCLC patients with unknown EGFR mutation status requires further investigation through related studies including a higher number of cases.

Anterior gradient 2 is correlated with EGFR mutation in lung adenocarcinoma tissues.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) has demonstrated a promising therapeutic response in lung adenocarcinoma patients with EGFR gene mutations. However, the predictive factors for this therapy have not been established, except for the EGFR gene mutation status of carcinoma cells. We first performed microarray analysis in EGFR-TKI-sensitive lung adenocarcinoma cell lines. The results indicated anterior gradient 2 (AGR2) as a potential surrogate marker of EGFR-TKI. Therefore, we then evaluated the correlation between the status of AGR2 immunoreactivity and clinicopathological factors including overall survival (OS), progression-free survival (PFS) and clinical response to EGFR-TKI, in 147 cases of surgically resected lung adenocarcinoma. The biological significance of AGR2 was further evaluated by transfecting small interfering RNA (siRNA) against AGR2 in these cells. The status of AGR2 immunoreactivity was significantly higher in lung adenocarcinoma cases with EGFR gene mutations than in those with the wild type (p<0.0001), but there were no significant differences in OS, PFS and response of EGFR-TKI between the AGR2 high and low carcinoma cases. Knockdown of AGR2 gene expression following siRNA transfection resulted in a significantly lower response to EGFR-TKI in EGFR-mutated PC-3. AGR2 could serve as an adjunctive surrogate protein marker possibly reflecting EGFR gene mutations in lung adenocarcinoma patients. Results from in vitro analysis indicated that AGR2 could be a potential clinical biomarker of EGFR-TKI therapeutic sensitivity in lung adenocarcinoma cells.