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

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.

Anaplastic lymphoma kinase gene rearrangement and non-small cell lung cancer management: a step forward in personalized therapy

Dear Editor, We greatly enjoyed reading the thought-provoking article by Lopes et al. in a recent issue of your esteemed journal (1). Interestingly, in recent years, data have emerged regarding a role for EML4 (echinoderm microtubule-associated protein-like 4)–ALK (anaplastic lymphoma kinase) in personalized treatment for advanced non-small-cell lung cancer (NSCLC) (2,3). EML4-ALK undergoes constitutive dimerization through the interaction between the coil-coil domains within the EML4 region of each monomer, thereby activating ALK and promoting oncogenic activity (4). EML4-ALK fusion, EGFR, and KRAS mutations are all mutually exclusive, implicating ALK rearrangement as a potential therapeutic target in EGFR wild-type and KRAS wild-type lung cancers (5). Nevertheless, Lopes' study (1) raises some interesting questions. Was patient selection for the study carried out appropriately? Did the study provide a representative sample of the Latin-American population, as described in the paper? Could immunohistochemistry be substituted for fluorescence in situ hybridization? Which technique is more appropriate for clinical practice? Should we perform this analysis in a specific subset of patients or in all advanced NSCLC patients? In addition, it is interesting to speculate whether EML4-ALK-targeted therapies may be relevant for clinicians, although patients positive for the EML4–ALK fusion protein frequently present at an advanced clinical stage, and their tumors demonstrate a solid adenocarcinoma pattern and signet ring cells (5). Furthermore, the presence of the EML4-ALK fusion oncoprotein is associated with nonsmokers or light smokers and is more frequent in younger patients (5). Thus, young, non-smoking patients and patients with adenocarcinoma tumor histology may indeed benefit from an EML4-ALK diagnostic test. Recently, the U.S. Food and Drug Administration approved crizotinib, which is a small-molecule inhibitor of the ALK tyrosine kinase, as a treatment for patients with locally advanced or metastatic NSCLC expressing the EML4–ALK fusion protein (2). The results were recently reported for a clinical trial in which 82 ALK-positive patients were evaluated for the therapeutic efficacy of crizotinib (6). The results were quite promising, demonstrating an overall response rate of 57% (46 partial responses and 1 complete response) and a 33% stable disease rate (27 patients) (2). Of the 82 patients, 63 (77%) continued to receive crizotinib at the time of data cut off, and the estimated probability of a 6-month progression-free survival was 72% (2). Thus, the refined understanding of the NSCLC molecular profile described over the last decade has proven to be an important tool to help medical oncologists develop new approaches for NSCLC treatment. Despite the small frequency of advanced NSCLC patients who present with EML4-ALK fusion (2.4 to 6.7%) (1), we believe that all patients in this setting should have the opportunity to receive such innovative therapies and approaches. However, further studies are warranted to establish the appropriate pharmaco-economic profiles.

CHFR aberrant methylation involves a subset of human lung adenocarcinoma associated with poor clinical outcomes

Excluding epidermal growth factor receptor (EGFR) mutation, v-Ki-ras2/Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion, the genetic alterations involved in lung adenocarcinogenesis, especially those linked to poor clinical outcomes, are still unknown. In this study, we analyzed abnormal checkpoint gene with forkhead-associated domain and ring finger (CHFR) methylation along with the above 3 mutations in 165 lung adenocarcinomas, evaluated the spectrum of each molecular abnormality, and correlated the results with clinical and pathologic variables. Reverse transcription-polymerase chain reaction assay, reverse transcription-polymerase chain reaction followed by direct DNA sequencing, and methylation-specific polymerase chain reaction were performed to detect these 3 mutations and CHFR hypermethylation. The EML4-ALK transcript or CHFR hypermethylation was found in 11 (6.7%) or 16 (10%) adenocarcinomas, respectively, whereas EGFR or KRAS mutation was detected in 48 (29%) or 13 (8%) cases, respectively. EGFR mutations occurred in patients who were negative for both CHFR hypermethylation and KRAS mutation. Among the 4 genetic or epigenetic abnormalities, only CHFR hypermethylation was significantly correlated with poor prognosis and lymphatic vessel invasion (P = .024). Histopathologically, the molecular abnormality that correlated with alveolar-destructive growth was the CHFR hypermethylation rather than the EGFR mutation (P = .03). Our results demonstrate that CHFR hypermethylation maybe one of the molecular abnormalities involved in a subset of lung adenocarcinomas with poor prognoses that might be induced by destructive growth and lymphatic vessel invasion of carcinoma cells. Thus, CHFR abnormality might be pursued as a novel therapeutic target against lung adenocarcinoma without an already-known mutation.

Histologic and Molecular Characterization of Lung Cancer With Tissue Obtained by Electromagnetic Navigation Bronchoscopy

Electromagnetic navigation bronchoscopy (ENB) is a catheter-based adjunct to standard bronchoscopic techniques for the sampling of lung lesions. We sought to evaluate the adequacy of ENB-obtained samples for histologic subtyping of lung cancer, epidermal growth factor receptor (EGFR) mutations, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocations. We retrospectively analyzed consecutive patients who underwent ENB for the diagnosis of lung lesions between 2008 and 2011. In those proven to be a primary lung cancer by ENB, tissue adequacy for histologic subtyping was recorded. Accuracy was determined by comparison with resected specimens when available. Tissue adequacy for EGFR mutation and/or EML4-ALK analyses was also reviewed. Sixty-five ENB cases resulted in a diagnosis of lung cancer. Tissues obtained were adequate for histologic subtyping in all 65 cases. Forty-three (66.2%) were diagnosed with adenocarcinoma, 19 (29.2%) with squamous cell carcinoma, 3 (4.6%) with small cell carcinoma. In 51 cases (78.5%), subtyping was performed by morphology alone, whereas 11 (21.5%) required immunohistochemical staining. Sixteen of 65 tumors underwent surgical resection. Concordance of histologic subtyping between ENB and surgical specimens was 87.5% (14 tumors). ENB-obtained samples from 15 patients with adenocarcinoma were sent for EGFR mutation analysis, of which 14 (93.3%) were adequate. Samples from 2 patients were evaluated for EML4-ALK gene rearrangements, both of which were adequate for analysis. ENB is effective at obtaining tissue samples adequate for histologic subtyping, EGFR mutation, and EML4-ALK translocation analysis.

Solitary pulmonary metastasis from lung cancer harboring EML4–ALK after a 15-year disease-free interval

It is often difficult to differentiate metachronous primary lung cancers from local pulmonary recurrences when the histopathological findings are similar. A 43-year-old man underwent right upper lobectomy with lymph node dissection for primary lung adenocarcinoma (p-T2aN0M0, stage IB). Fifteen years later, he developed a lung nodule in his right middle lobe. The tumor was preoperatively thought to be a metachronous second primary lung adenocarcinoma, and was surgically resected. Histopathological findings for both tumors were of poorly differentiated adenocarcinoma with mucus production. Both tumors also harbored the EML4 (echinoderm microtubule-associated protein-like 4)–ALK (anaplastic lymphoma kinase) fusion gene (variant 3a+b). Based on this molecular finding, the pulmonary nodule was considered to be a recurrence after very long latent period.

Histologic and Molecular Characterization of Lung Cancer With Tissue Obtained by Electromagnetic Navigation Bronchoscopy

Background: Electromagnetic navigation bronchoscopy (ENB) is a catheter-based adjunct to standard bronchoscopic techniques for the sampling of lung lesions. We sought to evaluate the adequacy of ENB-obtained samples for histologic subtyping of lung cancer, epidermal growth factor receptor (EGFR) mutations, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocations. Methods: We retrospectively analyzed consecutive patients who underwent ENB for the diagnosis of lung lesions between 2008 and 2011. In those proven to be a primary lung cancer by ENB, tissue adequacy for histologic subtyping was recorded. Accuracy was determined by comparison with resected specimens when available. Tissue adequacy for EGFR mutation and/or EML4-ALK analyses was also reviewed. Results: Sixty-five ENB cases resulted in a diagnosis of lung cancer. Tissues obtained were adequate for histologic subtyping in all 65 cases. Forty-three (66.2%) were diagnosed with adenocarcinoma, 19 (29.2%) with squamous cell carcinoma, 3 (4.6%) with small cell carcinoma. In 51 cases (78.5%), subtyping was performed by morphology alone, whereas 11 (21.5%) required immunohistochemical staining. Sixteen of 65 tumors underwent surgical resection. Concordance of histologic subtyping between ENB and surgical specimens was 87.5% (14 tumors). ENB-obtained samples from 15 patients with adenocarcinoma were sent for EGFR mutation analysis, of which 14 (93.3%) were adequate. Samples from 2 patients were evaluated for EML4-ALK gene rearrangements, both of which were adequate for analysis. Conclusions: ENB is effective at obtaining tissue samples adequate for histologic subtyping, EGFR mutation, and EML4-ALK translocation analysis.

Advances of molecular subtype and targeted therapy of lung cancer

随着肺癌发生、发展和预后相关分子机制研究的不断深入，肺癌靶向治疗取得了较大的进展，每一种分子分型的发现都会带来相应靶向药物的研究。2004年表皮生长因子受体（epidermal growth factor receptor, EGFR）基因在非小细胞肺癌中的发现，为我们带来了EGFR突变高度敏感有效的酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）。2007年棘皮类微管相关样蛋白-4-间变型淋巴瘤激酶（EML4-ALK）融合基因的出现为肺癌的分子发展带来了新的有效靶点。目前多个肺癌分子靶点及其靶向药物正在研究中。本文旨在回顾并总结肺癌分子分型及其靶向治疗的研究进展。

Adenocarcinoma of the lung with miliary brain and pulmonary metastases with echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase translocation treated with crizotinib: A case report

Few descriptions of miliary brain metastases have been reported. We report the case of an adenocarcinoma of the lung associated with metachronous miliary brain and lung metastases with an echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) gene translocation. The patient was treated with crizotinib and showed a twelve-month progression-free survival. Clinicians should be attentive of the evolution of brain metastases with patients presenting EML4-ALK translocation.

Personalized medicine and treatment approaches in non-small-cell lung carcinoma.

Chemotherapy has been the traditional backbone for the management of metastatic lung cancer. Multiple trials have shown the benefits of treatment with platinum doublets in lung cancer. This “one treatment fits all” approach was further refined by the introduction of targeted agents and discovery of subpopulations of patients who benefited from treatment with these agents. It has also become evident that certain histologic subtypes of non-small-cell lung cancer respond better to one cytotoxic chemotherapy versus others. This has led to the concept of using histology to guide therapy. With the introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and the discovery of activating mutations in the EGFR gene, further personalization of treatment for subgroups of patients has become a reality. More recently, the presence of a fusion gene, echinoderm microtubule-associated protein-like 4 – anaplastic lymphoma kinase (EML4-ALK), was identified as the driver mutation in yet another subgroup of patients, and subsequent studies have led to approval of crizotinib in this group of patients. In this article, efforts in personalizing delivery of care based on the histological subtypes of lung cancer and the role of K-RAS and EGFR mutations, EML4/ALK translocation, and ERCC1 (excision repair cross-complementing 1) and EGFR expression in choosing appropriate treatments for patients with advanced lung cancer are discussed. This article also reviews the problem of resistance to EGFR tyrosine kinase inhibitors and the ongoing trials that target novel pathways and mechanisms that are implicated in resistance.

Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing

Echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non-small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

Triple probe FISH approach for detecting EML4-ALK gene rearrangement in Chinese patients with NSCLC.

e21080 Background: The echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion represents a novel target for the therapy of a subset of non-small cell lung cancer (NSCLC). ALK rearrangement has been found in approximately 1.6–11.6% of NSCLC in unselected patient populations, but the frequency also varied when different diagnostic approach was used. We, for the first time, investigated frequency of EML4-ALK rearrangement in unselected Chinese patients with NSCLC using recent developed fluorescence in situ hybridization (FISH) triple color break-apart probe. Methods: One hundred-thirty one FFPE specimens from NSCLC patients were collected for the investigation. Fusion between 3’ portion of ALK gene and the 5’ portion of EML4 gene was detected using FISH tri-check probe (ZytoVision, Germany). Positive cells were defined as having any signal A ( distance more than two-time the ALK signal size) or signal B ( distance between one- and two-time the ALK signal size plus EML4 translocation signal) or any isolated ALK 3’ signal. Total 100 nuclei for each specimen were counted. Specimen was as classified as EML4-ALK rearrangement when the proportion of positive cells was over 15%. ALK/EML4 signals over 6 in over 10% nuclei were classified as gene amplification. Results: Of 131 specimens, frequency of ALK rearrangement was 10.7% (14/131) when positive cells were counted by signal A and isolated ALK 3’ signal (dual color). Frequency of EML4-ALK rearrangement was 13.0% (17/131) when positive cells were counted by signal A, B and isolated ALK 3’ signal (triple color), in which 21% (17/14) more cases were classified as EML4-ALK rearrangement comparing dual color enumeration. ALK and EML4 amplification were observed in 2.3% (3/131) specimens, respectively, in which two were co-amplified and none of them had EML4-ALK rearrangement. Conclusions: Present result showed that frequency of EML4-ALK rearrangement in unselected Chinese patients with NSCLC was 13.0% determined by FISH tri-check probe. This study suggested that counting both ALK inversion and EML4 translocation may contribute to defining EML4-ALK positive cells.

Crizotinib in the treatment of non-small-cell lung cancer

Introduction: Recent progress in identifying distinct subsets of lung cancer, based on critical driver mutations, has led to increasingly focused efforts in the development of selectively targeted therapies. The fusion oncogene, echinoderm microtubule-associated protein-like 4 – anaplastic lymphoma kinase (EML4-ALK), is present in approximately 5% of non-small-cell lung cancer (NSCLC) tumors. Crizotinib is an oral tyrosine kinase inhibitor (TKI), which silences the protein product of the ALK fusion gene and has recently been approved for the treatment of NSCLC aberrantly expressing ALK. Emerging data suggest that crizotinib may also have activity in other subsets of lung cancer, including tumors demonstrating amplification or mutation of the MET oncogene, or translocation of the ROS1 oncogene.Areas covered: This paper gives an overview of the molecular pathogenesis of ALK-associated NSCLC. It also reviews the pharmacokinetic and pharmacodynamic data on crizotinib and outlines the preclinical and clinical studies leading to the approval of crizotinib. In addition, it discusses its role in the treatment of NSCLC expressing ALK.Expert opinion: Crizotinib represents the newest example of a focused strategy for drug development in lung cancer, based on identification and targeted inhibition of critical tumor-specific driver mutations. Crizotinib has demonstrated efficacy against ALK-rearranged NSCLC, and has potential for broader application in select subsets of lung cancer.

Abstract IA2: Network models in oncogene-addicted lung cancer

Abstract Lung cancer is a devastating worldwide disease yet enthusiasm exists for treatment of subsets of the disease with molecularly targeted agents. Mutations in the epidermal growth factor receptor (EGFR) or translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) define two unique subsets of lung cancer characterized by sensitivity to tyrosine kinase inhibitors (TKI). Despite striking results with TKI, not all patients respond, the drugs are non-curative, and resistance is universal. Mutations in KRAS also define a group of patients awaiting therapeutic opportunities. We are characterizing signaling networks using tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions (PPI) and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. In PC9 cells with mutated EGFR, we characterized a physical EGFR network consisting of 266 proteins by integrating both TAP and pTyr MS data. In H3122 cells harboring EML4-ALK, we identified a PPI network consisting of 113 proteins and using pTyr MS identified changes in tyrosine phosphorylation in 120 proteins (58 decreased, 62 increased) following exposure to ALK tyrosine kinase inhibitor. Functional proteins are being discovered from these networks using siRNA and inhibitor screens. In parallel studies, we have exploited the use of chemical proteomics to discern targets of promiscuous kinase inhibitors and enable optimal combination approaches. In this way, we view network mapping linked to chemical proteomics as one approach to discern novel drug combination studies for in vivo validation and ultimately translation to early phase clinical trials. To translate PPI based mass spectrometry studies to clinic, we are developing in situ assays that identify and quantify PPI using proximal ligation assays (PLA). Pilot studies identify EGFR:Grb2 interactions in formalin fixed human lung cancers. These approaches have the potential to enable ‘network medicine’ by identifying novel combination approaches as well as through identifying subtypes of cancers through network views of cancer.

EML4-ALK Translocation Predicts Better Outcome in Lung Adenocarcinoma Patients with Wild-Type EGFR

The echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion represents a novel target in a subset of non-small cell lung cancer, especially adenocarcinoma. EML4-ALK fusion is mutually exclusive with epidermal growth factor receptor (EGFR) mutations. To understand the impact of EML4-ALK on the prognosis of non-small cell lung cancer, we examined EML4-ALK fusion in lung adenocarcinoma from patients with wild-type EGFR and analyzed their clinical treatment outcomes. Lung adenocarcinoma patients with malignant pleural effusions having wild-type EGFR and measurable target lesions were enrolled for EML4-ALK analysis by reverse transcription-polymerase chain reaction and direct sequencing. Demographic data, EML4-ALK status, and survival data were analyzed. We also performed fluorescence in situ hybridization on some available tumor samples to validate the PCR result. In addition, K-ras mutation was analyzed for patients without EML4-ALK fusion genes. A total of 116 patients with wild-type EGFR sequencing results had complete clinical data for analysis. No patients received ALK inhibitor therapy. There were 39 patients (34%) with the EML4-ALK fusion gene. The concordance rate between reverse transcription-polymerase chain reaction and fluorescence in situ hybridization was 85%. The K-ras mutation rate for patients without EML4-ALK fusion gene was 6.5%. By multivariate analysis, patients who had better performance status (p < 0.001) and EML4-ALK translocation (p = 0.017) had longer overall survival. Comparing patients with tumors harboring variant 1 with those harboring nonvariant 1 EML4-ALK fusion genes, there were no significant differences in clinical factors and survival outcome. For lung adenocarcinoma patients with wild-type EGFR, EML4-ALK translocation is associated with longer overall survival.

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.

Personalized Therapy for Non-Small Cell Lung Cancer: Which Drug for Which Patient?

The elucidation of the molecular alterations in non-small cell lung cancer (NSCLC) and the development of molecularly targeted agents have permanently shifted NSCLC therapy to a personalized approach. In the metastatic setting, the addition of the anti-vascular endothelial growth factor monoclonal antibody, bevacizumab, to chemotherapy improves overall survival. The oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, prolong progression-free survival in patients selected for the presence of an EGFR activating mutation. The monoclonal antibody to EGFR, cetuximab, improves survival in patients with metastatic NSCLC, and the inhibitor of the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein, crizotinib, has resulted in an unprecedented overall survival advantage in patients harboring the EML4-ALK translocation. In the adjuvant setting, gefitinib has not been shown to improve patient survival outcomes; however, there are several ongoing clinical trials in the adjuvant setting evaluating the role of erlotinib, bevacizumab, and the MAGE-A3 and MUC1 vaccines. The realm of personalized lung cancer therapy also includes the study of chemotherapy selected on the basis of the pharmacogenetic profile of a patient's tumor. Several ongoing clinical trials in both the metastatic and adjuvant settings are studying the excision repair cross-complementing group 1 (ERCC1) protein, the ribonucleotide reductase subunit 1 (RRM1) protein, thymidylate synthase, and BRCA1 as predictors of chemotherapy response. This review will outline the current state of the art of personalized NSCLC therapy.

Research progress in non-small cell lung cancer with concomitant EML4-ALK fusion gene and EGFR gene mutation

肺癌是最常见的恶性肿瘤之一，其中非小细胞肺癌（non-small cell lung cancer, NSCLC）占肺癌的80%-85%。分子靶向治疗是目前NSCLC最热门也是最具前景的领域之一，其中的热点分子包括表皮生长因子受体（epidermal growth factor receptor, EGFR）、棘皮动物微管样蛋白4-间变淋巴瘤激酶（echinoderm microtubule associated protein like4-anaplastic lymphoma kinase, EML4-ALK）等。既往研究认为EML4-ALK融合基因与EGFR突变不能共存。近期陆续报道了EML4-ALK融合基因与EGFR突变共存的病例。本文就EML4-ALK融合基因及EGFR突变基因的分子结构、发生率和目前已报道双突变患者的临床特点等进行综述。

Bright-Field Dual-Color Chromogenic In Situ Hybridization for Diagnosing Echinoderm Microtubule-Associated Protein-Like 4-Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinomas

A subset of lung cancers harbors an EML4-ALK (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase) gene fusion, and detecting this subset may hold therapeutic implications. Many prior studies used fluorescence in situ hybridization (FISH) analysis for this detection, but FISH may have disadvantages including signal decay and dark-field examination that may obscure tissue architecture. In this study, we explored the potential of the ALK-break-apart chromogenic in situ hybridization (CISH) method to detect ALK-rearranged lung cancer. We examined 15 lung adenocarcinomas with reverse-transcriptase polymerase chain reaction-proven EML4-ALK fusion transcripts and 30 ALK-negative cases. One hundred tumor cells were evaluated by CISH and FISH for each case, and a detailed signal profile was recorded and compared. CISH preserved tissue architecture and cytomorphology considerably and facilitated the signal evaluation using a routine light microscope. Positive rearrangement signals (splits or isolated 3' signals) were identified in 13 to 78% (mean ± SD, 41% ± 19%) of tumor cells in the ALK-positive cohort and in 0 to 15% (mean ± SD, 6% ± 4%) of cells in the ALK-negative cohort. The two groups were best separated by a cutoff value of 20%, with a sensitivity of 93% and a specificity of 100%. The only false-negative tumor having only 13% CISH-positive cells displayed predominantly (76%) isolated 5' signals unaccompanied by 3' signals. FISH showed largely similar signal profiles, and the results were completely concordant with CISH. We have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This method allows simultaneous visualization of genetics and tumor cytomorphology and facilitates the molecular evaluation and could be applicable in clinical practice to detect lung cancer that may be responsive to ALK inhibitors.

Abstract 1627: Proteome-wide analysis of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) network in lung cancer

Abstract The translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) defines a unique, new subset of patients with non small cell lung cancer (NSCLC). It represents a novel diagnostic biomarker and therapeutic target for NSCLC. PF 02341066 is a ALK small tyrosine kinase inhibitor that has demonstrated high clinical activity in EML4-ALK- positive NSCLC patients. We sought to characterize the signaling network used by EML4-ALK and reveal potential new therapeutic targets. We used tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. We selected the human NSCLC H3122 cell line for our studies as it is sensitive to PF 02341066 and harbors ELM4-ALK. Quantitative western blotting results show that activities of MET, ALK, AKT, ERK and STAT3 were inhibited by treatment with PF 02341066. Proteome-wide analysis of tyrosine phosphorylation of the ALK signaling pathway were examined. In total, 311 tyrosine phosphorylated proteins (487 unique phosphorylated sites) were identified. Of those, 73 pTyr sites (62proteins) were upregulated and 78 pTyr sites (58 proteins) were downregulated by 1uM PF02341066. Using TAP, we identified 13 proteints interacting with EML4-ALK including GRB2 and SHC1. Core interactions were verified by coupling of immunoprecipitation to Western blot and mass spectrometry. A comprehensive physical ALK network was generated by integrating both phosphoproteomic data and interactional partners using pathway analysis algorithms. Two adaptors, SHC1and GAB2, are suggested as the first outlets of signaling propagation from ALK. Other sublevels directly connected with ALK may allow further understanding the mechanism of ‘ALK addiction’. A sub-network composed of 35 cell death associated proteins was created. An ongoing functional analysis including siRNA and small inhibitor screen on this small network should validate druggable targets. This network approach has potential translational applications to identify novel approaches to treatment of ALK addicted lung cancers and could be useful in patients with acquired resistance to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2011-1627