Alk mutation analysis is used primarily to determine if a person with nonsmall cell lung cancer nsclc is likely to respond to an alk kinase inhibitor drug therapy, such as crizotinib. We aimed at revealing the clinical features of eml4alk fusion gene and egfr mutation in nonsmallcell lung cancer nsclc. The gene view histogram is a graphical view of mutations across eml4. Eml4alk fusion gene mutations in tissue samples and blood samples. The probe is designed by labeling the 3 and a 5 portions of the alk gene in different colors. Largescale screening and molecular characterization of eml4.
Comparison of clinicopathological features and prognosis. Droplet digital pcr ddpcr, a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of alk rearrangements and. Eml4alk fusion gene was analyzed by realtime polymerase chain reaction, and egfr mutations were analyzed by amplified refractory mutation system. Mutation analysis can help predict sensitivity or resistance to first and second generation inhibitors such as crizotinib, alectinib, and ceritinib. Representative expression profiles of selected samples using an alk fusion transcript assay. Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting eml4alk fusion protein, displays remarkable initial responses, a fraction of nsclc cases. Patients harboring alk rearrangements tend to be never and light smokers, have a history of. A novel mutation in the alk domain, encoding a g1269a amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Firstly, it doesnt discriminate between the variant eml4alk fusions. We also present the followup results of patients with multiple synchronous luac who had egfr mutation and eml4alk fusion gene.
Specification mutation for test generation and analysis. Upregulation of pdl1 by eml4alk fusion protein mediates. Clinical outcome for eml4alkpositive patients with advanced non. This testing detects the presence of alk gene rearrangements in tumor tissue. Alk eml4alk mutation is found in about 5 percent of people with nsclc. Lung cancer is the leading cause of cancerrelated death globally. The introduction of the eml4alk mutation in the cell line was confirmed via sanger sequencing as shown in figure 2 a, b for the expected 5p and 3p genomic breakpoints. Mechanisms of resistance to crizotinib in patients with. Eml4alk mutations in lung cancer that confer resistance to alk inhibitors. Alkpositive samples, as determined by fish and ihc.
The egfr mutations and the eml4alk fusion genes are coexistent. Driver genes as predictive indicators of brain metastasis. Further analysis revealed that eml4 alk was present at a frequency of 16. Analysis of somatic copy number aberrations scna demonstrated that a gain in eml4 was associated with longer pfs, and a loss of alk or gain in egfr was associated with shorter pfs. Nov 25, 2019 the univariate analysis showed that age p. Clinical significance of eml4alk fusion gene and association. Two patients, one with a resistance mutation, exhibited new onset alk copy number gain cng. The egfr, pik3ca, and kras assays require genomic dna. Arms showed 27 cases with eml4 alk fusion gene mutation, the mutation detection rate was 23. The egfrkras mutations and the eml4alk translocation are called driver mutations because they are responsible for both the initiation.
We report the 5th case which also did not show good response. Concomitant eml4alk rearrangement and egfr mutation in nonsmall cell lung cancer patients. To the best of our knowledge, five patients with both mutations have been reported so far in the world 58. Activity of egfrtyrosine kinase and alk inhibitors for eml4. The test is typically ordered along with or as a followup test to egfr and ros1. Identification of novel variant of eml4alk fusion gene in nsclc 2 march 2012 vol. In contrast, the patient in case 2, who had 2 tumors that originated from different clones with egfr mutation and eml4alk rearrangement, respectively, underwent only 6 cycles of platinumbased chemotherapy and chest radiotherapy, however, progressionfree survival pfs reached more than 1 year. The eml4alk echinoderm microtubuleassociated proteinlike 4 gene and the anaplastic lymphoma kinase gene fusion oncogene was recently identified as a novel genetic alteration in nonsmallcell lung cancer nsclc. About 47% of nonsmall cell lung carcinomas nsclc have eml4 alk translocations. In contrast, the patient in case 2, who had 2 tumors that originated from different clones with egfr mutation and eml4 alk rearrangement, respectively, underwent only 6 cycles of platinumbased chemotherapy and chest radiotherapy, however, progressionfree survival pfs reached more than 1 year.
Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. Analysis of realword mutations of lung cancer driver. In 20, tracheal, bronchus and lung tbl cancer has led 1. Droplet digital pcr for absolute quantification of eml4alk. The benefit of the alk fish is twofold, she explains. The positive rate of eml4alk mutation was calculated as 6. There was no statistical association between eml4alk mutation and either sex p0. Software installation the alk rearrangement kit has a designated kit app and. This abnormal gene fusion leads to the production of a protein eml4 alk that. May 29, 20 the eml4alk echinoderm microtubuleassociated proteinlike 4 gene and the anaplastic lymphoma kinase gene fusion oncogene represents a novel molecular target in a small subset of nonsmallcell lung cancers nsclcs. Nextgeneration sequencing was used to detect the mutation of eml4alk in 1505 nonsmall cell lung cancer patients, including 1208 tissue samples and 297 blood samples. The mutation of egfr and eml4alk was detected by realtime polymerase chain reaction rtpcr using the amplification refractory mutation system. Lung adenocarcinoma with concomitant egfr mutation and alk rearrangement.
The anaplastic lymphoma kinase alk gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. An overview of anaplastic lymphoma kinase alk anaplastic lymphoma kinase, which is a member of theinsulin receptor proteintyrosine kinase superfamily, is most closely related to leukocyte tyrosine kinase ltk 8. Targeting stemness is an effective strategy to control. Of note, 15 patients had double mutations in egfr and alk. Because the eml4alk fusion was only shown by a reverse transcriptionpolymerase chain reaction approach, we developed fluorescent in situ hybridization assays to. The mutation of egfr and eml4 alk was detected by realtime polymerase chain reaction rtpcr using the amplification refractory mutation system. Mutation analysis 23 is a method for developing sets of test cases which are sensitive to small syntactic structural changes. It is used to guide treatment for nsclc and other cancers results from the alk gene mutation analysis test may aid in the selection of therapeutic drugs, such as tyrosine kinase inhibitors tkis, and disqualify the usage of certain drugs. Eml4alk translocations in lung cancer quest diagnostics. Should everyone with lung cancer have alk mutation testing. Concomitant egfr mutation and eml4alk rearrangement in.
To date, there is only one published report from india on eml4 alk mutations demonstrating a positivity of 2. We proudly present gepia2, an updated and enhanced version of gepia. Multiplexed gene expression and fusion transcript analysis to. Analysis of egfr, eml4alk, kras, and cmet mutations in. Data analysis was performed with massarray typer software version 4. Concomitant eml4 alk rearrangement and egfr mutation in nonsmall cell lung cancer patients. Restrict the view to a region of the gene by dragging across the histogram to highlight the region of interest, or. Eml4alk testing in nonsmall cell carcinomas of the lung. Oct 16, 2008 a, stably expressed alk and its mutants f1174l and k1062m were immunoprecipitated with an antiflag antibody and subjected to western blot analysis with antipy1604 upper panel or py20 middle.
Nov 11, 2015 we aimed at revealing the clinical features of eml4 alk fusion gene and egfr mutation in nonsmallcell lung cancer nsclc. Eml4 alk positive lung cancer is a primary malignant lung tumor whose cells contain a characteristic abnormal configuration of dna wherein the echinoderm microtubuleassociated proteinlike 4 eml4 gene is fused to the anaplastic lymphoma kinase alk gene. The kit of claim 1, further comprising a primer pair comprising a forward primer and a reverse primer for amplifying a fragment of the nucleic acid sample which contains the eml4alk fusion mutation. The mutation rates of other genes were 18% eml4alk, 6% ros1, 5% her2. B, fluorescenceactivated cell sorting analysis of subg 1 fraction without treatment left column and after treatment with 0. The characteristics of the eml4 alk fusion gene in chinese patients with nsclc are poorly understood. Eml4alk fusions have been detected in 2 to 7% of nsclc patients. Analysis of egfr, eml4alk, kras, and cmet mutations in chinese lung adenocarcinoma patients article in experimental lung research 398 august 20 with 47 reads how we measure reads. Within this cohort, the eml4 alk fusion gene was identified in 12 tumors 11.
Comparison of eml4alk fusion gene positive rate in. Detection of eml4alk fusion gene and features associated with. Sanger sequencing of prepared eml4alk cdna from mrna of the mutated cell line was carried out to confirm the expression of the eml4alk fusion transcript figure 2c. Hip1alk, a novel alk fusion variant that responds to crizotinib. Jul 31, 2008 i got my tumour tested for the eml4 alk mutation by sending tissue to boston, and it turned out that i did have the mutation. Eml4 alk fusion gene was analyzed by realtime polymerase chain reaction, and egfr mutations were analyzed by amplified refractory mutation system. It tends to involve younger people and nonsmokers, or light. To aid in identification and treatment of these patients, we examined the clinical characteristics and treatment outcomes of patients who had nsclc with and without eml4alk. In 115 cases of nsclc, ihc showed 32 cases with alk d5f3 expression, the expression rate was 27. Mechanisms of resistance to crizotinib in patients with alk. Whereas the true frequency of eml4 alk fusion events remains to be determined, our fish results from two independent populationbased cohorts zurich and michigan, comprising more than 600 patients, suggest that the eml4 alk fusion may be less frequent 2. Chimeric proteins, such as eml4 alk, contain an nterminus derived from eml4 fused to the cterminal kinase domain of alk, which permits the ligandindependent dimerization and.
The eml4 alk fusion gene has been recently identified in a small subset of nonsmall cell lung cancer nsclc patients who respond positively to alk inhibitors. We also determined whether tae684, a specific alk kinase inhibitor, would inhibit the growth of eml4alk containing cell lines in vitro and in vivo. The transforming eml4alk fusion gene was first reported in nonsmall cell lung carcinoma in 2. Here, we report on the prevalence of eml4 alk, egfr status and kras mutations in 208 chinese patients with nsclc. Jul 11, 2007 pcr analysis of genomic dna from one of these two patients further revealed the breakpoints in eml4 and alk as well as the formation of both eml4alk and alkeml4 fusion genes, thus. Purpose the eml4alk fusion oncogene represents a novel molecular target in a small subset of nonsmallcell lung cancers nsclc. Alk inhibitors are potential anticancer drugs that act on tumours with variations of anaplastic lymphoma kinase alk such as an eml4 alk translocation. Nonsmall cell lung carcinoma nsclc accounts for approximately 85% of lung. Because of this profound therapeutic implication, the latest national comprehensive cancer network clinical practice guidelines. Jan 24, 2018 molecular testing for epidermal growth factor receptor egfr mutation and anaplastic lymphoma kinase alk and ros proto. It will detect any rearrangement of the alk gene because the probe uses the so called breakapart strategy. Patients and methods patients with nsclc were selected for genetic screening on. Ventana immunohistochemical staining of the eml4alk was performed. We experienced a rare case of the patient who had both egfr mutation and eml4 alk fusion gene.
The cells were treated with indicated concentrations of alk. Oncogenic mutations of alk kinase in neuroblastoma nature. Expanded circulating tumor cells from a patient with alkpositive. Rnaseq was conducted to further confirm the expression of eml4alk and tpm3ros1 fusions at. The incidence of eml4 alk fusion in egfr negative patients was 11. Driver genes as predictive indicators of brain metastasis in. Quantitative detection of alk fusion breakpoints in plasma cellfree. We determined the frequency of eml4alk in caucasian nsclc and in nsclc cell lines. Caliez j, monnet i, pujals a, rousseaubussac g, jabot l, boudjemaa a. Clinical features and outcome of patients with nonsmall. Driver mutations of young lung adenocarcinoma patients with. Detection of eml4alk fusion gene and features associated.
Characterization of alk fusion patterns and their resulting clinicopathological profiles could be of great benefit in better. Proposal for an external quality assessment program. These mutations are displayed at the amino acid level across the full length of the gene by default. Corresponding sequence chromatograms of samples are represented in a, b, and c, showing eml4alk v1, v2, and v3a, respectively. Gepia gene expression profiling interactive analysis. However, point mutations in the alk tyrosine kinase domain, such as those detected by this test, are reported in patients who develop resistance to this therapy. Kras mutations are involved about 25 percent of the time. Each operator is a pattern for a small syntactic change. About 47% of nonsmall cell lung carcinomas nsclc have eml4alk translocations approved inhibitors. The results showed that intron 5 of eml4 fused to intron18 of alk, and intron 7 of tpm3 was ligated to intron 34 of ros1. We aimed to determine appropriate diagnostic and therapeutic strategies for dualpositive patients with multiple synchronous luac. Correlation between eml4alk, egfr and clinicopathological f.
The results showed that intron 5 of eml4 fused to intron18 of alk figure 2a, and intron 7 of tpm3 was ligated to intron 34 of ros1 figure 2b. Alk phosphorylation in the compound mutation harboring cells was suppressed in ceritinib and brigatinib. Two cases showed good response 5, 6, whereas the other two cases did not. Four patients 36% developed secondary mutations in the tyrosine kinase domain of alk. The fusion between anaplastic lymphoma kinase alk and echinoderm microtubuleassociated proteinlike 4 eml4 is a causative factor in a unique subset of patients with nonsmall cell lung carcinoma nsclc. Patients with anaplastic lymphoma kinase alk gene rearrangements often manifest dramatic responses to crizotinib, a smallmolecule alk inhibitor. Anaplastic lymphoma kinase gene alk fusions have been identified in approximately 5% of nonsmallcell lung carcinomas nsclcs and define a distinct subpopulation of patients with lung cancer who are highly responsive to alk kinase inhibitors, such as crizotinib. However, it was not correlated with the gender, smoking history, maximal. A case of lung adenocarcinoma harboring egfr mutation and. Fusion partners of alk include npm, eml4, tpm3, atic, tfg, cars, and cltc. Correlation between eml4alk, egfr and clinicopathological. In particular, eml4alk fusions that are commonly observed in.
Full text clinical and epidemiological study of egfr. Upregulation of pdl1 by eml4alk fusion protein mediates the. I started the trial 6 weeks ago, and 2 weeks ago i had a second pet scan which showed that in only 4 weeks there had been a significant reduction in both size and activity. Ventana immunohistochemical staining of the eml4 alk was performed. Users can perform all expression analyses such as survival analysis and differential analysis at the isoform level. The gepia server has been running for two years and processed 280,000 analysis requests for 110,000 users from 42 countries. The positive rate of eml4 alk mutation was calculated as 6. Concomitant egfr mutation and eml4alk rearrangement in lung. Eml4alk fusion gene and efficacy of an alk kinase inhibitor. Only one case each of concurrent egfr or kras mutation was detected. In this study, mutational status of egfr and gene fusions in eml4alk was examined extensively in multiple synchronous luac from 1059 surgical resection luac.
We enrolled 694 chinese patients with nsclc for analysis. Nov 27, 2019 to further study the specific fusion site of eml4alk and tpm3ros1 at dna level, sequencing reads were examined on integrative genomic viewer igv software. Fusion of eml4 and alk is associated with development of lung. Restrict the view to a region of the gene by dragging across the histogram to highlight the region of interest, or by using the sliders in the filters panel to the left.
Multiplexed gene expression and fusion transcript analysis. Mutation detection is based on allelespecific amplification and detection with hydrolysis probes. Eml4alk positive lung cancer is a primary malignant lung tumor whose cells contain a characteristic abnormal configuration of dna wherein the echinoderm microtubuleassociated proteinlike 4 gene is fused to the anaplastic lymphoma kinase gene. Overcoming resistance by alk compound mutation i1171s. Crizotinib treatment significantly prolongs progressionfree survival, increases response rates, and improves the quality of life in patients with alkpositive nonsmallcell lung cancer. Alk mutation analysis is used primarily to determine if a person with adenocarcinoma nonsmall cell lung cancer is likely to respond to an alk kinase inhibitor drug therapy, such as crizotinib. Identification of novel variant of eml4alk fusion gene in. Frontiers genomic profiling reveals synchronous bilateral. Sample negative for alk rearrangement by fish but positive for alk expression by ihc. Alk mutation gene rearrangement lab tests online au. The mutation of eml4alk in the tissue samples of 399 patients with nonsmall cell lung cancer was detected by reverse transcription polymerase chain reaction. The eml4alk assay and the lung cancer rna panel require total rna input and are based on.