EML4-ALK Fusion Transcripts Research Articles

Molecular characteristics and clinical outcomes of complex ALK rearrangements identified by next-generation sequencing in non-small cell lung cancers

BackgroundComplex kinase rearrangement, a mutational process involving one or two chromosomes with clustered rearrangement breakpoints, interferes with the accurate detection of kinase fusions by DNA-based next-generation sequencing (NGS). We investigated the characteristics of complex ALK rearrangements in non-small cell lung cancers using multiple molecular tests.MethodsSamples of non-small cell lung cancer patients were analyzed by targeted-capture DNA-based NGS with probes tilling the selected intronic regions of fusion partner genes, RNA-based NGS, RT-PCR, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).ResultsIn a large cohort of 6576 non-small cell lung cancer patients, 343 (5.2%) cases harboring ALK rearrangements were identified. Fourteen cases with complex ALK rearrangements were identified by DNA-based NGS and classified into three types by integrating various genomic features, including intergenic (n = 3), intragenic (n = 5) and “bridge joint” rearrangements (n = 6). All thirteen cases with sufficient samples actually expressed canonical EML4-ALK fusion transcripts confirmed by RNA-based NGS. Besides, positive ALK IHC was detected in 13 of 13 cases, and 9 of 11 cases were positive in FISH testing. Patients with complex ALK rearrangements who received ALK inhibitors treatment (n = 6), showed no difference in progression-free survival (PFS) compared with patients with canonical ALK fusions n = 36, P = 0.9291).ConclusionsThis study firstly reveals the molecular characteristics and clinical outcomes of complex ALK rearrangements in NSCLC, sensitive to ALK inhibitors treatment, and highlights the importance of utilizing probes tilling the selected intronic regions of fusion partner genes in DNA-based NGS for accurate fusion detection. RNA and protein level assay may be critical in validating the function of complex ALK rearrangements in clinical practice for optimal treatment decision.

P28-11 The CRISPR-Cas13a gene-editing system underlies a potential therapeutic strategy for EML4-ALK-positive lung cancer cells

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer, and the majority of these are adenocarcinoma. One of the most common types of oncofusion in lung adenocarcinoma is EML4-ALK which leads to the constitutive activation of ALK and thus increased cell survival, cell growth, and proliferation. Hence, treatment with an ALK tyrosine kinase inhibitors provoke a significant anti-tumor response; however, they inevitably succumb to the acquired resistance. Therefore, an alternative therapeutic strategy that limits ALK over activation is necessary for the treatment of this lung cancer. The existence of EML4-ALK fusion oncogene in H3122 and H2228 lung cancer cells had been confirmed using direct sequencing. Next, the expression of both genes had been observed using the qRT-PCR. Subsequently, the EML4-ALK fusion transcripts were knocked down by using the CRISPR-Cas13a effector tilling three guide RNAs. The knockdown effect on the cell physiology was then validated by cell viability, apoptosis indicator markers, and phosphorylation experiments. Based on the expression results, the EML4 transcript was not substantially expressed but ALK expressed 100 fold and 80 fold higher in H3122 and H2228 cells separately compare to a non-fusion transcript in HEK293T cells. The EML4-ALK transcripts were significantly down-regulated by employing Cas13a in those lung cancer cells. Consequently, EML4-ALK knockdown considerably inhibited the lung cancer cell viability as well as induced cell death. Furthermore, the phosphorylation is reduced up to seventy percent after the knockdown in ALK. Overall the obtained data demonstrated that EML4-ALK transcript knockdown using CRISPR-Cas13a inhibited EML4-ALK-positive lung cancer cell survival and proliferation. This study suggests that CRISPR-Cas13a mediated EML4-ALK RNA knockdown devises an effective therapeutic strategy for treating ALK-rearranged NSCLC patients.

EML4-ALK fusion transcripts identification in circulating blood platelets and exosomes in non-small cell lung cancer patients

EML4-ALK fusion transcripts identification in circulating blood platelets and exosomes in non-small cell lung cancer patients

Localized Single Transcript Detection of Eml4‐Alk in NSCLC using Co‐localization Quantum Dot Fluorescent In Situ Hybridization (CoQFISH)

Lung cancer remains one of the most common forms of cancer worldwide with more than 80% being non-small cell lung cancer (NSCLC). Two primary clinical tests are used to identify NSCLC subtypes through the detection of EGFR mutation and oncogenic gene fusions such as EML4-ALK. Since the discovery of EML4-ALK, there has been a multitude of targeted therapies such as ALK inhibitors alectinib, ceritinib, and crizotinib, which has shown great success in treatment. However, the methods for clinical detection of gene fusions remains constant through either RT-PCR or Break Apart FISH. By observing co-localization of signal in oligonucleotide conjugated quantum dots, we designed a novel co-localization based quantum dot fluorescent in situ hybridization (CoQFISH) technology capable of detecting sensitively and specifically single localized fusion transcripts using as little as two oligonucleotide probes in cell lines and tissue. We found that CoQFISH can detect EML4-ALK transcripts in a positive control NSCLC cell line H2228 using the co-localization of Eml4 and Alk targeted probe sets, which was verified through qPCR and targeted RNA-seq. Furthermore, we found that CoQFISH can positively identify EML4-ALK transcripts in human NSCLC tissue biopsies at a sensitivity that is higher than qPCR and RNA sequencing and offer information on the spatial location of each transcript. Looking further, quantum dot probes in CoQFISH are uniquely suited for resolving smaller areas on transcripts for identification of targets such as gene isoform transcripts. This novel technology would not only aid in research on understanding fusion genes and small RNA transcripts, but more importantly allow more sensitive diagnostics of potential cancer targets meaning earlier detection and better therapeutic response monitoring. Detection of CoQFISH probes targeting GAPDH. Positive signal was detected in human lung tissue for the positive control containing GAPDH (left). Human tissue was treated with RNAse treatment in the negative control (right) and showed no detected signal. CoQFISH detects Eml4-Alk fusion transcripts. Differential interference contrast (DIC) shows human non-small cell lung cancer tissue structure on the right. Co-localized signal from Eml4 (middle) and Alk (right) is indicated with the corresponding arrows. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 1739: Analysis of EML4-ALK fusion transcripts in plasma and platelets to monitor response to crizotinib in EML4-ALK positive non-small cell lung cancer patients (NSCLC)

Abstract Background: Rearrangements in anaplastic lymphoma kinase (ALK) gene can be detected in 5-7% of EGFR and KRAS wild-type advanced NSCLC patients (p). Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently used for screening but are unable to identify the specific fusion partner and are unpractical to monitor clinical responses due to difficulty of obtaining rebiopsies. The RT-PCR technique has the potential to overcome this pitfall and allow patient monitorization in blood. Methods: A total of 405 formalin-fixed paraffin-embedded (FFPE) samples from advanced NSCLC were analyzed by ALK IHC (Ventana D5F3) and FISH (Vysis). Positive patients were confirmed by RT-PCR and submitted to Sanger in order to identify the variant. In a subset of 36 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, fusion transcripts were analyzed by RT-PCR in mRNA purified from plasma and platelets and correlated with clinical response. Results: ALK IHC was analyzed in 405 NSCLC patients and 37 tested positive (9.1%) whereas 25 (7.7%) were identify as translocated by FISH (n=323). ALK fusion transcripts were analyzed by RT-PCR and a new fusion variant of ALK was identified. A total of 36 p benefited from crizotinib treatment, including the p with the new variant. Monitoring of EML4-ALK fusion transcripts in the plasma ad platelets of 35 ALK positive patients revealed a good correlation with clinical outcome to crizotinib treatment, with the fusion transcripts becoming undetectable in p with good clinical responses. Conclusions: Analysis of ALK fusion transcripts in mRNA purified from plasma and platelets can have a value in patients with no biopsy available and to monitor the course of the disease. Citation Format: Cristina Aguado, Cristina Teixido, Ana Gimenez-Capitan, Maria de los Llanos Gil, Sonia Rodriguez, Santiago Viteri, Niki Karachaliou, Erika Aldeguer, Vicente Peg, Lidia Alonso, Miguel Angel Molina-Vila, Rafael Rosell. Analysis of EML4-ALK fusion transcripts in plasma and platelets to monitor response to crizotinib in EML4-ALK positive non-small cell lung cancer patients (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2017-1739

P3.02a-008 EML4-ALK in Plasma Exosomes from a Cohort of NSCLC Patients: Topic: ALK Biomarkers

The identification of EML4-ALK translocations in 3%-5% of NSCLC resulted in the approval of numerous targeted therapies of these lung cancers. Consequently, the availability of new ALK inhibitors forced the development of diagnostic assays, although their validation is limited by the restricted number of available specimens. As the significance of the current standard test (tissue-based FISH) is progressively questioned, development of improved accompanying diagnostics for the predictive biomarker is of high medical need. Overcoming discrepancies of FISH such as tissue sample quality and inter-observer variability, we validated a liquid biopsy test for analysis of EML4-ALK fusion transcripts. For the first time, the availability of a defined cohort of NSCLC patients allowed the determination and clinical interpretation of EML4-ALK in exosomal RNA derived from plasma specimen. We developed and validated an exosome-based biopsy test for EML4-ALK to be run in a CLIA laboratory. The assay enables the discriminative detection of the three major fusion variants v1, v2 and v3 (a,b,c). In detail, exosomal RNA is isolated from low-volume plasma (≤ 2ml) of NSCLC patients and subjected to highly sensitive and specific RT-qPCR. During analysis, each sample is confirmed by defined test controls and process tracking. The final diagnostic results of all clinical specimen are correlated with clinical response data. Section is not applicable. Cohort analysis is ongoing. Therefore, we will submit our results as late-breaking abstract. Section not applicable. We will determine the variant-specific expression of EML4-ALK in plasma samples of a clinically defined, medium-sized NSCLC cohort. Based on specimen data, we are able to test for correlation of the biomarker derived from exosomes with respective tissue results and clinical response of a patient.

Abstract 857: Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC

Abstract Clinical testing for the detection of RNA fusions in tissue currently include FISH, IHC, PCR and NGS. However, approximately 30% of patients with advanced non-small cell lung cancer (NSCLC) are not candidates for tissue biopsies and in some cases where tissue is obtained, it is not always of sufficient quantity for molecular testing. For these reasons, the detection of nucleic acids in circulation has become increasingly relevant to clinical testing. In this study, we have focused on the development of an EML4-ALK diagnostic test system that includes the prospective collection of whole blood and the reproducible detection of mRNA fusion transcripts by a PCR-based technology. The focus was on the detection of EML4-ALK transcripts from donors with and without previously diagnosed NSCLC. Pre-analytic complexity was reduced by restricting the handling of samples to 72 hours from time of sample receipt to test result. Recovery methods for RNA extraction from donor plasma were then optimized to enrich for RNA recovered from both circulating-free, and RNA within blood vesicles, including platelets and exosomes. A two-step reverse transcription and Droplet Digital™ PCR (RT-ddPCR) method was evaluated in detection testing using a multiplexed EML4-ALK assay that includes variants 1, 2 and 3. Analytic assay specificity and sensitivity was examined for RT-ddPCR efficiency using a cell-line positive for EML4-ALK variant 1 and in vitro RNA designed to mimic variants 1, 2, and 3. Analytic sensitivity was determined to be 0.2% of fusion RNA spiked into a background of normal plasma. Precision studies were conducted with two different amounts of input RNA (high and low), over three consecutive days, three runs in one day and with two operators. The SD of detected fusion transcripts in this study did not exceed 25%. Finally, normal (n = 10) and FISH positive NSCLC donor (n = 9) plasma samples were processed for the recovery of RNA and tested for EML4-ALK fusions. All FISH positive cases were accurately detected by the RT-ddPCR test with EML4-ALK variant copies ranging from 13 to 150 copies. EML4-ALK fusion transcripts were not identified in normal donor plasma. We conclude that the developed test system is highly suited for a 72 hour test to result turnaround, reproducible and sensitive detection of diagnostic fusion RNA variants, including EML4-ALK in blood in the clinical laboratory. Citation Format: Hestia Mellert, Leisa Jackson, Dianna Maar, Dawne Shelton, Gary Pestano. Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 857.

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer.

Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK- platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.

Simple and Rapid In Vivo Generation of Chromosomal Rearrangements using CRISPR/Cas9 Technology

Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk-rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo.

TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC

ObjectivesLung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). Materials and methodsThis assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). ResultsEML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. ConclusionOur variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.

Abstract 336: Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy.

Abstract Background: Recent studies have characterized non-small cell lung cancer (NSCLC) as one of the most genomically deranged of all cancers, necessitating that both new drug development and patient therapy account for intra- and inter-patient tumor heterogeneity. Clinically annotated NSCLC patient-derived xenograft (PDX) models represent a novel approach to integrate this genomic complexity into a clinically relevant pre-clinical platform. We here describe molecular characterization to profile all currently “druggable” oncogenes for NSCLC in paired PDXs and original patient NSCLC tumor (PT). Method: Genomic DNA from archival formalin-fixed, paraffin-embedded (FFPE) PT and fresh first human-to-mouse (P0) NSCLC PDX tumors were isolated and subjected to oncogene mutational profiling using Sequenom's OncoCarta Panel v1. This panel detects 238 mutations in 19 genes commonly altered in cancer. RT-PCR-based molecular analyses of EGFR and KRAS mutations, EML4-ALK fusion transcripts, and RNA expression levels of ERCC1, RRM1 and TS genes were performed by Response Genetics, Inc. Genomic DNA from 3 serially passaged NSCLC PDX tumors (2 KRAS and 1 EGFR mutation models) up to 5 passages were also analyzed. Results: In the first 7 of 9 patient-PDX NSCLC models tested, oncogene mutational fidelity was preserved between PDX and PT with a good correlation of molecular biomarker expression (p<0.01). Two paired models had discrepancies in genotyping: from harboring 2 or 3 oncomutations at a frequency of 5-17% in PT to no mutation detected in P0 tumors), likely due to intra-patient tumor heterogeneity from clonal evolution. Of 3 models that have serial passaged tumors, the frequencies of oncomutation in each model were similar among the same passage (P0) or serial passage (P0 to P5) tumors. In several models tested for in vivo drug efficacy based on the molecular biomarker expression, results matched treatment outcome of the original patients., Conclusion: Our results validate the overall genomic fidelity of PDX tumors compared to original PT. Molecular characterization of individual tumor results in a clinically and genomically annotated PDX model with potential utility for selecting and validating clinically relevant drug target(s) for personalized cancer therapy. Acknowledgement: Supported by UC Davis Comprehensive Cancer Center Developmental Award (NIH/NCI P30CA093373), UL1 RR024146 from the National Center for Research Resources, the Jackson Laboratory, Response Genetics Inc., and the Addario Foundation. Citation Format: Sonal J. Desai, Neal Goodwin, Regina Gandour-Edwards, Royce F. Calhoun, David T. Cooke, Laurel A. Beckett, Martin K.H. Maus, Stephanie H. Astrow, Philip C. Mack, Ralph deVere White, David R. Gandara, Tianhong Li. Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2013-336

Update on the large-scale screening of ALK fusion oncogene transcripts in archival NSCLC tumor specimens using multiplexed RT-PCR assays.

7594 Background: The ALK inhibitor crizotinib offers a new standard of care for advanced NSCLC patients with EML4-ALK fusion oncogenes. We previously reported a 4.0% frequency of EML4-ALK fusion oncogene transcripts detected in 1889 NSCLC specimens in the RGI database (Li et al., ASCO 2011). Methods: Patented single and multiplexed RT-PCR assays suitable for rapid and accurate detection of all variants of ALK fusion oncogene transcripts were used as previously described, including all 9 known EML4-ALK fusion gene transcripts and ALK RNA levels (Danenberg, ASCO 2010). The sensitivity and specificity on archival formalin-fixed, paraffin-embedded tumor specimens are 99% and 100%, respectively. We here update the detection of EML4-ALK fusion transcripts in the RGI database. Results: Between 12/2009 and 09/2011, 4750 NSCLC specimens in the RGI database were tested for the presence of ALK fusion transcripts. We found 152 (3.2%) NSCLC cases with EML4-ALK fusion positivity, including 87 (57.2%) V1, 15 (9.9%) V2, 47 (30.9%) V3, and 3 (2.0%) V5a variants. Median age (range): 61.1 (33-96). Female: 74 (49%). All EML4-ALK-positive tumors were adenocarcinomas. No EGFR or K-Ras mutation was detected in ALK fusion-positive samples. Expression of chemotherapy-related biomarkers was available from 63 (female: 31, 49%) EML4-ALK-positive cases in the database: 43 (68%) had low TS level of <2.33; 40 (63.5%) had low ERCC1 level of <1.7, and 25 (40%) had low RRM1 level of <0.97. Conclusions: This RT-PCR assay provides a tool for rapid, large-scale screening of NSCLC FFPE tissues for EML4-ALK fusion gene transcripts. The relative value of this RT-PCR assay as a companion diagnostic test for drugs targeting ALK merits evaluation in comparison with the FDA approved ALK FISH test.

Abstract 3745: Detection of various ALK translocations using intragenic differential expression (IDE) in patients with non-small cell lung cancer

Abstract Introduction: Efforts to target EML4-ALK fusions with ALK inhibitors in non-small cell lung cancer (NSCLC) have shown promising results in patients harboring a paracentric inversion on chromosome 2, inv(2)(p21p23). In effect, the clinical utility of these drugs is dependent on the presence of ALK gene activation. Reliable testing for ALK activation by translocation is important for selecting patients for this therapy. Although 11 variants for translocation are known, new variants have recently been reported. The aim of this study was to develop a reliable molecular assay to detect translocation of the ALK gene irrespective of the breakpoint on the EML4 gene or the partner gene. To achieve this, we used intragenic differential expression (IDE) of ALK gene comparing expression levels of the 5′ end with the 3′ end. Methods: ALK IDE was determined by measuring the levels of both 5′ and 3′ transcript regions by quantitative RT-PCR. IDE scores were obtained by calculating the endogenous control (ABL1) normalized differences in 5′ and 3′ ALK levels (IDE = 5′ALK/ABL1 - 3′ALK/ABL1). High IDE score, relative to normal, indicates the presence of ALK rearrangement. Relative expression of ALK (independent of rearrangement) was also established. A subset of samples were also analyzed by fluorescence in situ hybridization (FISH). All study samples were analyzed for direct detection of EML4-ALK by RT-PCR performed in a parallel study. Results: The ALK IDE value of an EML4-ALK fusion-positive cell line (NCI-H2228) was 0.7 whereas it was 0.0 for 2 EML4-ALK-negative NSCLC cell lines (NCI H838 and NCI H1299). The positive control value (0.7) was set as the cutoff to distinguish positive vs. negative ALK rearrangement. Eleven percent (6/56) of the lung cancer tissue samples were IDE positive. Eighty-three percent (5/6) of these positives were confirmed by direct detection of EML4-ALK fusion transcript by RT-PCR, including one specimen harboring the previously undescribed variants 8a and 8b. Five IDE-positive samples and 5 with slightly-to-moderately elevated levels of ALK transcript (3′ ALK > 0.1) were further analyzed by FISH. Of these 10 samples, 80% (8/10) showed ALK rearrangement and/or gene amplification. All samples interpreted as having ALK rearrangements by FISH were also positive by IDE (3/3). Two IDE positive (1 confirmed, 1 unconfirmed by RT-PCR) were interpreted as rearrangement negative by FISH. Conclusions: ALK IDE accurately categorized all FISH-confirmed rearrangements as positive and detected rearrangements in at least 1 other confirmed case not identified by FISH. This method is useful for detection of EML4-ALK rearrangements and may function as a universal molecular assay for determining ALK rearrangements in multiple tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3745.

EML4-ALK fusion transcripts in immunohistochemically ALK-positive non-small cell lung carcinomas

EML4-ALK fusion transcripts have been found in a subset of non-small cell lung carcinomas (NSCLCs); however, their protein expression status has not yet been fully elucidated. In this study we investigated ALK protein expression in 302 NSCLCs and 291 gastric carcinomas by means of immunohistochemical analysis. Twelve (4.0%) NSCLCs, but none of the gastric carcinomas, were found to be positive for ALK. The ALK signal was detected in the cytoplasm of cancer cells. Subsequent RNA analysis of 10 RNA-available, immunohistochemically ALK-positive tumors revealed that three tumors had EML4-ALK variant 1, three tumors had variant 2, three tumors had variants 3a and 3b, and one tumor had a novel variant in which exon 14 of EML4 is connected to the nucleotide at position 53 of exon 20 of ALK by a 2-bp insertion. These results suggest that immunohistochemical ALK detection is a useful way to screen NSCLCs for tumors containing ALK fusions.

Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non–small cell lung carcinomas

Recently, the fusion gene EML4-ALK was identified in non-small cell lung carcinoma, which could be a potential therapeutic target. We investigated the prevalence of anaplastic lymphoma kinase protein expression in these tumors by immunohistochemistry and correlated the results with data from ALK molecular studies. Gene expression profiling was performed on 35 adenocarcinomas to identify cases with ALK gene up-regulation, which was correlated with protein overexpression by immunohistochemistry. Immunohistochemistry was also performed on an independent cohort consisting of 150 adenocarcinomas and 150 squamous cell carcinomas to evaluate the utility of anaplastic lymphoma kinase immunostaining as a screening tool. Florescence in situ hybridization for the ALK locus and reverse transcriptase-polymerase chain reaction for EML4-ALK were performed on tumors positive for anaplastic lymphoma kinase by immunohistochemistry. Transcriptional up-regulation of ALK was identified in 2 (6%) of 35 adenocarcinomas by gene expression profiling. These 2 cases were positive for anaplastic lymphoma kinase by immunohistochemistry, whereas the remaining 33 cases were completely negative. In the independent cohort, anaplastic lymphoma kinase immunostaining was positive in 1 of 150 squamous cell carcinomas and in 3 of 150 adenocarcinomas. The 6 cases positive for anaplastic lymphoma kinase by immunohistochemistry showed evidence of ALK locus rearrangement by florescence in situ hybridization but were negative for EGFR and KRAS mutation. The presence of EML4-ALK fusion transcript was confirmed in 2 cases by reverse transcriptase-polymerase chain reaction. In conclusion, anaplastic lymphoma kinase immunoreactivity in non-small cell lung carcinomas was associated with transcriptional up-regulation, ALK locus rearrangement, and the presence of EML4-ALK fusion transcript. Anaplastic lymphoma kinase immunohistochemistry may have utility as a screening tool or as a surrogate marker for the molecular techniques to detect the EML4-ALK fusion gene in these tumors.

Multiplex Reverse Transcription-PCR Screening for EML4-ALK Fusion Transcripts

EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non-small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes. Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs. From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens. These data reinforce the importance of accurate diagnosis of EML4-ALK-positive tumors for the optimization of treatment strategies.

EML4–ALK fusion transcript is not found in gastrointestinal and breast cancers

Fusion genes have been identified as chromosomal rearrangements in certain cancers, such as leukaemia, lymphoma, and sarcoma. The EML4–ALK (EML4: echinoderm microtubule-associated-protein-like 4; ALK: anaplastic lymphoma kinase) fusion gene has been identified as an oncogene in non-small-cell lung cancer (NSCLC). This study examined the presence of this fusion transcript in gastrointestinal and breast cancers. We evaluated the expression of the EML4–ALK transcript in 104 lung cancer cases and in 645 gastrointestinal and breast cancer samples. Only one of the lung cancer samples tested positive for the EML4–ALK fusion transcript, whereas none were detected in 555 gastrointestinal and 90 breast cancer cases. Our data suggest that the EML4–ALK fusion transcript is not present in gastrointestinal or breast cancers and is specific to NSCLC.

EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas

EML4-ALK gene fusions have recently been discovered in a subset of human lung carcinomas, and fusions of the ALK tyrosine kinase gene with the NPM, TPM3, CLTC, ATIC, and TFG genes have been found in hematological malignancies. To elucidate the role of fusions between ALK and other genes in pulmonary carcinogenesis, we examined 77 non-small cell lung carcinomas (NSCLCs) for EML4-, NPM-, TPM3-, CLTC-, ATIC-, and TFG-ALK fusion transcripts by RT-PCR and subsequent sequencing analysis. Although no expression of NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts were detected in any of the cases, expression of EML4-ALK fusion transcripts was detected in two (2.6%) of the 77 NSCLCs. In one of the two NSCLCs there was fusion between exon 13 of EML4 and exon 20 of ALK, i.e., variant 1, and in the other there was fusion between exon 20 of EML4 and exon 20 of ALK, i.e., variant 2. Both patients had a history of smoking, and histologically the carcinomas were adenocarcinoma. No somatic mutations were detected in the mutation cluster regions of the EGFR, K-RAS, and PIK3CA genes in these two carcinomas, however, a Pro177Ser mutation of the p53 gene was detected in the carcinoma that contained the variant 1 EML4-ALK fusion transcripts. In situ PCR of a paraffin block section showed that the carcinoma with expression of the variant 1 actually contained an EML4-ALK fusion gene. These results suggested that the EML4-ALK fusion gene product is involved in the carcinogenesis of a subset of NSCLCs.

Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.