Echinoderm Microtubule-associated Protein-like 4-anaplastic Lymphoma Kinase Fusion Gene Research Articles

Distribution of EML4-ALK fusion variants and clinical outcomes in patients with resected non-small cell lung cancer

ObjectivesThe molecular profiles and prognosis of anaplastic lymphoma kinase (ALK) fusion and resectable non-small cell lung cancer (NSCLC) remain unclear. This study aimed to explore the distribution of ALK fusion variants and prognostic factors in patients with surgically resected NSCLC. Material and methodsAmong the 93 ALK positive surgical patients screened by immunohistochemistry (IHC) or real-time polymerase chain reaction (RT-PCR), 63 patients were confirmed as ALK rearrangement by next-generation sequencing (NGS), including 55 cases of stage I-III and 8 cases of stage IV. Medical records were retrospectively reviewed, the distribution of ALK fusion variants and prognostic factors were analyzed. ResultsAll of the 55 early stage patients were histological adenocarcinoma. No other fusion types were found except for echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK). EML4-ALK variant 1 (E13:A20; 25/55, 45.5 %) was the predominant variant type, followed by EML4-ALK variant 3 (E6:A20; 19/55, 34.5 %) and variant 2 (E20:A20; 8/55, 14.5 %). Concomitant mutations occurred in 22 patients (22/55, 40.0 %), which involved in 32 co-mutations from 12 kinds of mutated genes. TP53 mutations were most common in coexisting mutations (13/32, 40.6 %). TP53 mutations were less frequently occurred in variant 1 group (3/25, 12.0 %) than in non-variant 1 group (10/30, 33.3 %, P = 0.064). The median disease-free survival (DFS) of the 55 patients was 22.1 months, and the median overall survival (OS) was not mature at the time of analysis. Multivariable analysis showed that stage T3 and EML4-ALK variant 3 were independent prognostic factors for shorter DFS. Neither TP53 mutations nor any coexisting mutations were related to prognosis. ConclusionsThis study illustrated the patterns of EML4-ALK fusion variants and gene profiles in patients with resected NSCLC. Advanced T stage and EML4-ALK variant 3 were associated with worse prognosis. The role of TP53 mutations in prognosis is worthy of further study.

Clinicopathological and Prognostic Significance of EML4-ALK Rearrangement in Patients with Surgically Resected Lung Adenocarcinoma: A Propensity Score Matching Study.

ObjectiveThe echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is a key oncogenic driver in non-small cell lung cancer (NSCLC). This study analyzed the clinicopathological characteristics and prognostic significance of EML4-ALK fusion gene in patients with surgically resected adenocarcinoma.MethodsThe clinicopathological characteristics of 1056 consecutive patients with surgically resected stage I–IIIA adenocarcinoma were collected from February 2014 to October 2014, and EML4-ALK rearrangement was detected using real-time polymerase chain reaction (RT-PCR) technology. To compare the imaging and pathological features, a propensity score matching (PSM) method was performed. The follow-up information was collected to evaluate the long-term outcomes of patients with EML4-ALK rearrangement.ResultsThe prevalence of EML4-ALK rearrangement was 6.6% in 1056 consecutive patients. A total of 70 EML4-ALK-positive and 210 EML4-ALK-negative patients were identified after PSM. Imaging and pathological analyses showed that EML4-ALK rearrangement was significantly associated with less ground-glass opacity (GGO) (adjusted OR=1.38, 95% CI=1.03–1.85, Ptrend=0.029) and higher prevalence of non-invasive mucinous adenocarcinoma mucin-laden adenocarcinomas (non-IMA MLA, adjusted OR=6.79, 95% CI=2.69–17.17, P<0.001). EML4-ALK rearrangement was found to be an unfavorable prognostic factor for disease-free survival (DFS) in female patients (HR=2.26, 95% CI=1.13–4.53, P=0.021).ConclusionOur results suggest that adenocarcinomas harboring EML4-ALK fusion gene exhibit specific radiological and pathological characteristics compared with EML4-ALK-negative adenocarcinomas. In female patients, EML4-ALK rearrangement was associated with shorter DFS.

Apatinib reverses alectinib resistance by targeting vascular endothelial growth factor receptor 2 and attenuating the oncogenic signaling pathway in echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene-positive lung cancer cell lines.

Overexpression of insulin growth factor 1 receptor (IGF-1R) and its ligand, insulin growth factor 1 (IGF-1), is related to treatment resistance and worse prognosis in many types of tumors. We reported recently that IGF-1R activation by IGF induces resistance to alectinib and stimulates the production of vascular endothelial growth factor, which indicates that IGF induces alectinib resistance and angiogenesis. This study aimed to determine the effect of bigeminal inhibition of anaplastic lymphoma kinase (ALK) and angiogenesis on human insulin growth factor 1 receptor (hIGF-1)-triggered drug resistance in echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK)-positive lung cancer. Human lung adenocarcinoma H3122 and H2228 cells were exposed to a combination of insulin growth factor 1 receptor (IGF-1), alectinib, or apatinib. The effects of the combination therapy were examined using cell the Cell Counting Kit-8 assay, the colony-forming assay, the scratch test, and flow cytometry analysis, and the molecular mechanism was assessed by western blot. At nontoxic concentrations, apatinib restored alectinib sensitivity by increasing drug-induced apoptosis and inhibiting viability, migration, and invasion in IGF-triggered drug resistant cells. Moreover, we found that apatinib restored sensitivity to alectinib mainly through suppression of the ALK downstream signaling pathway and antiangiogenesis signaling. Taken together, our results indicate that simultaneous inhibition of ALK and vascular endothelial growth factor R2 by the combination of alectinib with apatinib may be useful for controlling progression of EML4-ALK fusion gene lung cancer by reversing ALK-TKI resistance and inhibiting angiogenesis.

Clinical outcome study of crizotinib in immunohistochemistry-proven echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene among Indian patients with adenocarcinoma lung.

Aims:The anaplastic lymphoma kinase (ALK) Break Apart FISH Probe Kit and Ventana anti-ALK (D5F3) CDx immunohistochemistry (IHC) assay are the Food and Drug Administration-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The aim of this study was to assess the efficacy and safety of twice daily crizotinib tablet (250 mg) in IHC-proven echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene among Indian patients with adenocarcinoma lung in the routine clinical practice.Subjects and Methods:Patients with nonsmall cell lung cancer (NSCLC), adenocarcinoma histology, whose tumors were found to be positive for EML4-ALK fusion gene using IHC, were considered for this study. IHC analysis was performed using a Ventana automated immunostainer (Benchmark XT). Detection was performed using Optiview DAB detection and amplification kit.Results:A total of 25 NSCLC adenocarcinoma patients were included in the study. There were 14 (56%) women and 10 (44%) men with a median age of 53 years. All patients had Stage IV disease at the time of initiation of crizotinib therapy. One patient achieved complete response and 20 achieved response rate (PR) for an overall PR of 84%. The median progression-free survival (PFS) was 11.8 months and median overall survival (OS) was 20.6 months. Two (8%) patients experienced severe hepatotoxicity requiring permanent discontinuation of crizotinib therapy.Conclusions:A very high PR, PFS, and OS achieved in our study population indicates that IHC can accurately identify EML4 ALK fusion gene mutations in lung adenocarcinoma patients who are responsive to ALK inhibitors such as crizotinib. IHC should be considered as a cost-effective alternative to FISH, especially in low-resource countries.

Clinicopathological and molecular epidemiological study of lung cancer patients seen at a tertiary care hospital in Northern India.

Aims:The primary objective of this study was to estimate the clinicopathological and molecular profile of lung cancer patients along with the evaluation of their clinical characteristics at a tertiary care hospital in Northern India.Subjects and Methods:A total of 421 patients with lung cancer histology who were treated at Max Super Speciality Hospitals were included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and permission was obtained from the Ethics Committee before the start of the study. Clinical characteristics and molecular profiling data were collected from the patient's medical records.Results:There were 330 (78.4%) men and 91 (21.6%) women with a median age of 62 years (range: 30–93 years). Of the 421 patients, 388 (92.2%) patients had the nonsmall cell lung cancer (NSCLC) histology whereas 33 (7.8%) patients were of SCLC histology. Histology and gender had a significant association with NSCLC and SCLC (P < 0.05). Epidermal growth factor receptor (EGFR) and echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene testing was done in 120 and 93 patients, respectively. Of the 120 patients, 24 (20%) cases were positive for EGFR mutations whereas EML4-ALK fusion gene was present in 8 (8.6%) out of 93 patients.Conclusions:Our study confirms the importance of molecular testing in the NSCLC patient subgroup with an aim to identify the exact molecular targets that can benefit from the newer generation of targeted therapies.

Gene Expression and Clinical Characteristics of Molecular Targeted Therapy in Non-small Cell Lung Cancer Patients in Shandong

背景与目的分子生物学靶向治疗已逐渐成为非小细胞肺癌（non-small cell lung cancer, NSCLC）的一个重要治疗手段，本研究通过分析山东地区NSCLC多种驱动基因表达情况及临床病理特征，为筛选分子靶向治疗目标人群提供理论依据。方法采用荧光探针PCR法检测表皮生长因子受体（epidermal growth factor receptor, EGFR）、棘皮动物微管相关蛋白4-间变性淋巴瘤激酶（echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase, EML4-ALK）、肉瘤致癌因子-受体酪氨酸激酶（ROS proto-oncogene 1, receptor tyrosine kinase, ROS1）、鼠类肉瘤病毒癌基因（Kirsten rat sarcoma viral oncgene, KRAS）基因表达情况，回顾性分析阳性病例的临床病理特征。结果EGFR基因突变阳性率为36.70%，主要为19、21外显子突变，突变人群主要为女性、腺癌、不吸烟患者，组间差异有统计学意义。EML4-ALK融合基因重排阳性率为9.37%。人群特征主要为60岁以下不吸烟人群，组间差异有统计学意义，基因突变与病理类型和性别间无明显差异。ROS1融合基因重排阳性率为3.67%，均为60岁以下患者，组间差异有统计学意义。23份病例标本开展KRAS基因检测，阳性标本数2例，阳性率为8.70%。2份阳性标本均为60岁以上病例，男女各占1例，病理类型均为腺癌，均无吸烟史。此外，未发现有两种基因同时突变的病例。结论EGFR、EML4-ALK、ROS1、KARS基因在NSCLC患者中存在较高的突变率，且具有不同的人群特征，在选择靶向治疗人群中具有重要意义。

Detection of EML4-ALK fusion gene and features associated with EGFR mutations in Chinese patients with non-small-cell lung cancer.

PurposeEchinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC).MethodsWe enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system.ResultsAmong the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ2 test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations.ConclusionEML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent.

EML4-ALK translocation is associated with early onset of disease and other clinicopathological features in Chinese female never-smokers with non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocation is resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, but responds to the ALK-TKI crizotinib. Characterization of EML4-ALK translocation may provide invaluable information to facilitate disease diagnosis and improve the outcome of customized treatment. Although the occurrence of EML4-ALK translocation is likely to be affected by the smoking habits and gender of patients, the translocation has not been characterized extensively in female never-smokers with NSCLC. Therefore, 280 female never-smokers that were diagnosed with NSCLC were enrolled in the present study, and characteristics of EML4-ALK translocation, including the frequency, were determined in these NSCLC patients. EML4-ALK fusion variants were detected using Multiplex one-step reverse transcription-polymerase chain reaction and subsequently confirmed by DNA sequencing and Vysis ALK Break Apart fluorescence in situ hybridization analysis. The EML4-ALK fusion variants were detected in 21 carcinoma tissue specimens, accounting for 7.5% of the enrolled patients. Out of these patients with EML4-ALK fusion variants, EML4-ALK fusion variant 1 was identified in 12 patients, indicating that variant 1 is the most common type of EML4-ALK fusion gene in the present cohort of patients. ALK mRNA was aberrantly expressed in all the tissues with EML4-ALK translocation, but not in the carcinoma tissues without EML4-ALK translocation. In addition, the EML4-ALK translocation was more frequently found in younger patients. The median age of patients with EML4-ALK translocation was 50.95±2.29 years, which was significantly younger (P<0.01) than the median age of the patients without EML4-ALK translocation (57.15±0.56). The EML4-ALK translocation was detected exclusively in undifferentiated tumors that were graded as poorly- or moderately-differentiated carcinomas and suspected to be more malignant compared with well-differentiated tumors. In summary, the present study found that 7.5% of patients with NSCLC that are female never-smokers harbor EML4-ALK translocations, which are associated with the aberrant expression of ALK mRNA, early onset of disease and undifferentiated carcinomas.

Frequency of EGFR Mutation and EML4-ALK fusion gene in Arab Patients with Adenocarcinoma of the Lung

Abstract Introduction: Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. The frequency of epidermal growth factor receptor (EGFR) mutations is ethnicity-dependent, with a higher proportion in Asian populations than in whites, while the incidence of EML4-ALK (echinoderm microtubule-associated-protein like 4-anaplastic lymphoma kinase) fusion gene ranged from 1.6% to 16.4% in patients with NSCLC and these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. This study was conducted to determine the frequency of EGFR mutation and EML4-ALK fusion gene in our population and to determine the effect of different clinicopathological features on the expression of those mutations in patients with lung adenocarcinoma. Results: EGFR mutations were detected in approximately 33% of our patients in this series; the most frequently detected mutation was exon 19 deletion. EML4-ALK fusion gene was detected in 7.3% of patients. Conclusion: Our population exhibited the incidence of EGFR mutation approximately similar to that reported in East Asia and Japanese patients, higher than that recorded in USA, and Australia. However, more studies with larger patients’ numbers are needed to verify this finding.

Anaplastic lymphoma kinase translocation is correlated with anaplastic lymphoma kinase expression and mutually exclusive with epidermal growth factor receptor mutation in Taiwanese non-small cell lung cancer.

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is an important biomarker for target therapy. The aim of this study is to better understand the clinical and molecular features of the EML4-ALK fusion gene in lung cancer patients in Taiwan and therefore to generate an efficient algorithm for the detection of ALK translocation. In the first cohort, ALK translocation was identified in 1 adenocarcinoma from 100 lung cancer patients by using break apart fluorescent in situ hybridization (FISH). Next, we detected 6 ALK translocations in another 40 EGFR wild type adenocarcinomas but not in 40 cases with EGFR mutation. Histological analysis revealed that solid growth with signet-ring cells or cribriform glands with extracellular mucin were noted in all the 7 ALK translocated cases. One ALK positive cancer with mucinous cribriform pattern had no ALK expression. ALK expression was correlated with ALK translocation (p < 0.001), but not with ALK gene copy number gain (CNG) (P = 0.838). ALK translocation was also mutually exclusive with EGFR mutation in Taiwanese non-small cell lung cancer (P = 0.033). These results indicate that screening tests for EGFR mutation status and/or ALK expression could help efficiently select ALK translocated patients for target therapy.

Response to erlotinib in a patient with lung adenocarcinoma harbouring the EML4-ALK translocation: A case report.

Lung cancer is the leading cause of cancer-associated mortality worldwide, and the mainstay of treatment remains to be personalised therapy. Tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKIs) have been reported to exert a significant impact in the treatment of non-small cell lung cancer (NSCLC), particularly in patients harbouring mutations in the EGFR gene. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene translocation has been described in a subset of patients with NSCLC and possesses potent oncogenic activity. This translocation represents one of the most novel molecular targets in the treatment of NSCLC. Patients who harbour the EML4-ALK rearrangement possess lung tumours that lack EGFR or K-ras mutations. The present study reports the case of a patient possessing the EML4-ALK rearrangement that was initially treated with erlotinib and achieved a lasting clinical response. To the best of our knowledge, the current study is the first report of a clinical response to EGFR-TKI in a patient with lung adenocarcinoma harbouring the EML4-ALK fusion gene, but no EGFR mutations. However, as the disease progressed, the ALK gene status of the tumour was investigated, and based upon a positive result, the patient was treated with crizotinib and achieved a complete response. In conclusion, the present study suggests that the EML4-ALK rearrangement is not always associated with resistance to EGFR-TKIs. Further studies are required to clarify the biological features of these tumours and to investigate the mechanisms underlying the primary resistance to EGFR-TKIs when the EML4-ALK rearrangement is present.

Echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase rearrangement and epidermal growth factor receptor mutation coexisting in Chinese patients with lung adenocarcinoma.

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) rearrangement is almost in mutual exclusion to epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, with rare exceptions. This study aimed to search for the coexisting gene alterations in Chinese patients with lung adenocarcinoma (LAC). We detected mutations in the EGFR, KRAS, and ALK gene rearrangements in samples from 131 Chinese patients with LAC. ALK rearrangements were identified by fluorescent in situ hybridization. Mutations in EGFR (exons 19 to 21) and KRAS (codons 12 and 13) were determined by real time polymerase chain reaction. All patients were classified into four distinct genotype groups: EGFR mutations (n = 63; 48.1%), ALK rearrangements (n = 9; 6.9%), KRAS mutations (n = 8; 6.1%), and the wild-type (unmutated) genotype of all three genes (WT/WT/WT) (n = 53; 40.5%). Interestingly, two never-smoking women (2/131, 1.5%) harbored coexisting ALK rearrangement and EGFR mutation. ALK rearrangement occurred more frequently in young patients (8/9) (P = 0.687), non-smokers (8/9) (P = 0.077), and those who had no family history of LAC (8/9) (P = 1.000); all KRAS mutations occurred in the EGFR wild type (P = 0.007). KRAS mutations were generally detected in young patients (6/8) (P = 0.658) and in those who had no family history (7/8) (P = 1.000); EGFR mutations correlated with gender (P = 0.001), and smoking status (P < 0.001). Two patients harboring both EGFR mutation and EML4-ALK rearrangement were detected in this study. Our data was apparently inconsistent with the traditional view that the EML4-ALK fusion gene in patients is resistant to EGFR-TKIs.

Recurrence of Lung Adenocarcinoma After an Interval of 15 Years Revealed by Demonstration of the Same Type of EML4–ALK Fusion Gene

We carried out an experiment on a 58-year-old man with multiple left lung tumors and swelling of multiple lymph nodes. For clinical staging and therapeutic purposes, bronchoalveolar lavage (BAL) cytology and lung biopsy were performed. The biopsy specimen revealed the left lower lung mass to be immunohistochemically ALK (anaplastic lymphoma kinase)-positive adenocarcinoma. Using the BAL specimen from the left lower lung, EML4 (echinoderm microtubule-associated protein-like 4)-ALK variant 1 fusion gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). His past history showed that he had undergone an operation for lung adenocarcinoma of the right lower lobe 15 years before, and the pathological specimen at that time revealed that the lung adenocarcinoma with pleural invasion and single metastasis of mediastinal lymph node showed a mucinous cribriform pattern and/or signet-ring cell pattern. The typical histology led us to examine the ALK rearrangement in the primary lung cancer and mediastinal metastatic tumor. Immunohistochemistry (IHC) for ALK was positive, and ALK break apart fluorescence in situ hybridization (FISH) showed a positive result. Moreover, RT-PCR using formalin-fixed, paraffin-embedded tissue from the right lung cancer also demonstrated EML4–ALK variant 1 fusion gene. Although there is a possibility that the left lung cancer is de novo one with multiple metastases, detection of the same fusion gene of the very rare EML4–ALK variant 1 in both tumors suggests that the left cancer is a recurrence of the right lung cancer after an interval of 15 years.

Serum carcinoembryonic antigen levels before initial treatment are associated with EGFR mutations and EML4- ALK fusion gene in lung adenocarcinoma patients.

Epidermal growth factor receptor (EGFR) mutations and echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) define specific molecular subsets of lung adenocarcinomas with distinct clinical features. Our purpose was to analyze clinical features and prognostic value of EGFR gene mutations and the EML4-ALK fusion gene in lung adenocarcinoma. EGFR gene mutations and the EML4-ALK fusion gene were detected in 92 lung adenocarcinoma patients in China. Tumor marker levels before first treatment were measured by electrochemiluminescence immunoassay. EGFR mutations were found in 40.2% (37/92) of lung adenocarcinoma patients, being identified at high frequencies in never-smokers (48.3% vs. 26.5% in smokers; P=0.040) and in patients with abnormal serum carcinoembryonic antigen (CEA) levels before the initial treatment (58.3% vs. 28.6%, P=0.004). Multivariate analysis revealed that a higher serum CEA level before the initial treatment was independently associated with EGFR gene mutations (95%CI: 1.476~11.343, P=0.007). We also identified 8 patients who harbored the EML4-ALK fusion gene (8.7%, 8/92). In concordance with previous reports, younger age was a clinical feature for these (P=0.008). Seven of the positive cases were never smokers, and no coexistence with EGFR mutation was discovered. In addition, the frequency of the EML4-ALK fusion gene among patients with a serum CEA concentration below 5 ng/ml seemed to be higher than patients with a concentration over 5 ng/ml (P=0.021). No significant difference was observed for time to progression and overall survival between EML4-ALK-positive group and EML4-ALK-negative group or between patients with and without an EGFR mutation. The serum CEA level before the initial treatment may be helpful in screening population for EGFR mutations or EML4-ALK fusion gene presence in lung adenocarcinoma patients.

Analysis of clinicopathological features of the echinoderm microtubule-associated protein-like-4-anaplastic lymphoma kinase fusion gene in Chinese patients with advanced non-small-cell lung cancer.

The echinoderm microtubule-associated protein-like-4-anaplastic lymphoma kinase (EML4-ALK) fusion gene defines a novel molecular subset of non-small-cell lung cancer (NSCLC). However, the clinicopathological features of patients with the EML4-ALK fusion gene have not been defined completely. Clinicopathological data of 200 Chinese patients with advanced NSCLC were analyzed retrospectively to explore their possible correlations with EML4-ALK fusions. The EML4-ALK fusion gene was detected in 56 (28.0%) of the 200 NSCLC patients, and undetected in 22 (11.0%) patients because of an insufficient amount of pathological tissue. The median age of the patients with positive and negative EML4-ALK was 48 and 55 years, respectively. Patients with the EML4-ALK fusion gene were significantly younger (P< 0.001). The detection rate of the EML4-ALK fusion gene in patients who received primary tumor or metastatic lymph node resection was significantly higher than in patients who received fine-needle biopsy (P= 0.003). The detection rate of the EML4-ALK fusion gene in patients with a time lag from obtainment of the pathological tissue to EML4-ALK fusion gene detection ≤48 months was significantly higher than in patients >48 months (P= 0.020). The occurrence of the EML4-ALK fusion gene in patients with wild-type epidermal growth factor receptor (EGFR) was significantly higher than in patients with mutant-type EGFR (42.5% [37/87] vs. 6.3% [1/16], P= 0.005). Younger age and wild-type EGFR were identified as clinicopathological characteristics of patients with advanced NSCLC who harbored the EML4-ALK fusion gene. The optimal time lag from the obtainment of the pathological tissue to the time of EML4-ALK fusion gene detection is ≤48 months.

Comparison of IHC, FISH and RT-PCR Methods for Detection of ALK Rearrangements in 312 Non-Small Cell Lung Cancer Patients in Taiwan

BackgroundRecently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain.MethodsReverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed.ResultsThirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian.ConclusionsAmong the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

Analysis of EGFR, EML4-ALK, KRAS, and c-MET mutations in Chinese lung adenocarcinoma patients

ABSTRACTIntroduction: Mutation analysis of cancer driver genes is helpful for determining an optimal treatment strategy. We evaluated mutations in four driver genes, namely epidermal growth factor receptor (EGFR), Kirsten ras oncogene (KRAS), c-MET, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), in Chinese lung adenocarcinoma patients from Hunan Province. Methods: We enrolled 110 lung adenocarcinoma patients in a single institution. EGFR and KRAS mutations were examined by direct sequencing, the EML4-ALK fusion gene was analyzed by fluorescence in situ hybridization, and c-MET amplification and c-Met protein expression were detected by quantitative PCR and immunohistochemistry, respectively. Results: EGFR and KRAS mutations were observed in 52.7% (58/110) and 3.6% (4/106) of patients, respectively. c-MET amplification was detected in 5.5% (6/110) of patients. In addition, 30% (33/110) of the cases expressed c-Met protein, including all of the patients harboring c-MET amplification. Ten percent (11/110) of patients harbored the EML4-ALK fusion gene, and the frequency of ALK rearrangement was higher than that of other cohort analyses involving patients from other regions in China. Almost all of these gene mutations were exclusive except that in two female non-smoking patients, who harbored an EGFR mutation and EML4-ALK rearrangement simultaneously. In total, 70% of patients in the study harbored one of the four gene mutations. Conclusions: Most Chinese lung adenocarcinoma patients harbor driver gene mutations, among which ALK rearrangements were more common in Hunan patients than in previously reported populations. Future clinical trials should be conducted to determine the safety and efficacy of drug combination targeting different driver mutations.

A case of lung adenocarcinoma harboring exon 19 EGFR deletion and EML4-ALK fusion gene

We report a man with advanced adenocarcinoma who harboring exon 19 (E746-A750del) epidermal growth factor receptor (EGFR) deletion and echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) gene translocation in the re-biospy specimen. The patient was treated with erlotinib with a stable disease but progressed slowly, while crizotinib showed a complete response.

Membrane Phospholipids, EML4-ALK, and Hsp90 as Novel Targets in Lung Cancer Treatment

Approximately one third of patients with non-small cell lung cancer have unresectable stage IIIA or stage IIIB disease; combined cytotoxic chemotherapy and radiation therapy delivered concurrently has been established as the standard treatment for such patients. Despite many clinical trials that tested several different radiochemotherapy combinations, it seems that a plateau of efficiencies at the acceptable risk of complications has been reached. Clinical studies indicate that the improved efficacy of radiochemotherapy is associated with the radiosensitizing effects of chemotherapy. Improvement of outcomes of this combined modality by developing novel radiosensitizers is a viable therapeutic strategy. In addition to causing cell death, ionizing radiation also induces a many-faceted signaling response, which activates numerous prosurvival pathways that lead to enhanced proliferation in the endothelial cells and increased vascularization in tumors. Radiation at doses used in the clinic activates cytoplasmic phospholipase A2, leading to increased production of arachidonic acid and lysophosphatidylcholine. The former is the initial step in the generation of eicosanoids, while the later is the initial step in the formation of lysophosphatidic acid, leading to the activation of inflammatory pathways. The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) is member of the insulin superfamily of receptor tyrosine kinases. The EML4-ALK fusion gene appears unique to lung cancer and signals through extracellular signal regulated kinase and phosphoinositide 3-kinase. Heat shock protein 90 (Hsp90) is often overexpressed and present in an activated multichaperone complex in cancer cells, and it is now regarded as essential for malignant transformation and progression. In this review we focus on radiosensitizing strategies involving the targeting of membrane phospholipids, EML4-ALK, and Hsp90 with specific inhibitors and briefly discuss the combination of radiation with antivascular agents.

Correlation of anaplastic lymphoma kinase overexpression and the EML4-ALK fusion gene in non-small cell lung cancer by immunohistochemical study

Recently the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene with transforming activity was identified in non-small cell lung cancer (NSCLC). In addition, NSCLC patients with the EML4-ALK fusion gene had a dramatic response and longer progression free survival after ALK inhibitor treatment than those without this fusion gene. However, the incidence and clinical and molecular characteristics of the EML4-ALK fusion gene in NSCLC patients of Taiwan are still unclear. Sixty-four fresh frozen tumor specimens were obtained from the tissue bank of Chang Gung Memorial Hospital for RNA extraction and EML4-ALK fusion gene detection. Paraffin sections of lung tumors from all of these patients were available and were analyzed for ALK protein expression by immunohistochemical (IHC) study. The results were correlated with clinical and molecular biomarkers. Three of the 64 tumors (4.7%) had the EML4-ALK fusion gene. Two were adenocarcinomas, and one was adenosquamous carcinoma. Twenty patients with non-squamous cell carcinomas had epidermal growth factor receptor (EGFR) mutations, so the EML4-ALK fusion gene was found in 14.3% of EGFR wild type non-squamous cell carcinomas. Two tumors were variant 3 (3a+3b with 3b predominant) and had strong staining (3+) for ALK by IHC stains. One tumor was variant 1 and had moderate staining (2+) for ALK. None of the ALK wild type tumors had strong staining for ALK. When compared with other clinical and molecular features, only the IHC stain for ALK was significantly correlated with the EML4-ALK fusion gene (p = 0.0002). ALK overexpression detected by IHC study could be a promising detection method for the EML4-ALK fusion gene and is worth further confirmation with more samples.