EML4-ALK Fusion Research Articles

The repertoire of genetic alterations in salivary duct carcinoma including a novel HNRNPH3-ALK rearrangement

Salivary duct carcinoma (SDC) is a rare, aggressive malignancy with limited treatment options and poor outcome. Twenty-nine primary resected SDC, including 15 SDC de novo (SDCDN), and 14 SDC ex pleomorphic adenoma (SDCXPA) were subjected to the massive parallel sequencing assay (MSK-IMPACT) targeting 287 to 468 cancer-related genes. TP53 was the most frequently altered gene (69%). TP53 mutations and ERBB2 amplification were more frequent in SDCXPA than in SDCDN (P = .0007 and P = .01, respectively). Potentially targetable mutations were detected in 79% (23/29) of SDC involving ERBB2 (31%), PIK3CA (28%), HRAS (21%), ALK (7%) and BRAF (3%), and 22% (5/23) of those cases harbored possible primary resistance mutations involving CCNE1, NF1 and PTEN. A novel HNRNPH3-ALK rearrangement was found in one SDCDN. In another case, EML4-ALK fusion detected in the primary tumor was associated with ALK G1202R secondary resistance mutation in the post-treatment metastasis. A germline analysis of the DNA repair genes revealed a case with a pathogenic BRCA1 E23fs germline variant. SDCDN and SDCXPA are genetically distinct. Although the majority of SDC may be amenable to molecular targeted therapy, concurrent possible resistance mutations may be found in a significant minority of cases. A broad genomic profiling is necessary to ensure detection of rare but clinically actionable somatic alterations in SDC.

A combined gene expression tool for parallel histological prediction and gene fusion detection in non-small cell lung cancer

Accurate histological classification and identification of fusion genes represent two cornerstones of clinical diagnostics in non-small cell lung cancer (NSCLC). Here, we present a NanoString gene expression platform and a novel platform-independent, single sample predictor (SSP) of NSCLC histology for combined, simultaneous, histological classification and fusion gene detection in minimal formalin fixed paraffin embedded (FFPE) tissue. The SSP was developed in 68 NSCLC tumors of adenocarcinoma (AC), squamous cell carcinoma (SqCC) and large-cell neuroendocrine carcinoma (LCNEC) histology, based on NanoString expression of 11 (CHGA, SYP, CD56, SFTPG, NAPSA, TTF-1, TP73L, KRT6A, KRT5, KRT40, KRT16) relevant genes for IHC-based NSCLC histology classification. The SSP was combined with a gene fusion detection module (analyzing ALK, RET, ROS1, MET, NRG1, and NTRK1) into a multicomponent NanoString assay. The histological SSP was validated in six cohorts varying in size (n = 11–199), tissue origin (early or advanced disease), histological composition (including undifferentiated cancer), and gene expression platform. Fusion gene detection revealed five EML4-ALK fusions, four KIF5B-RET fusions, two CD74-NRG1 fusion and three MET exon 14 skipping events among 131 tested cases. The histological SSP was successfully trained and tested in the development cohort (mean AUC = 0.96 in iterated test sets). The SSP proved successful in predicting histology of NSCLC tumors of well-defined subgroups and difficult undifferentiated morphology irrespective of gene expression data platform. Discrepancies between gene expression prediction and histologic diagnosis included cases with mixed histologies, true large cell carcinomas, or poorly differentiated adenocarcinomas with mucin expression. In summary, we present a proof-of-concept multicomponent assay for parallel histological classification and multiplexed fusion gene detection in archival tissue, including a novel platform-independent histological SSP classifier. The assay and SSP could serve as a promising complement in the routine evaluation of diagnostic lung cancer biopsies.

Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non–Small Cell Lung Cancer in the Global Phase III ALEX Study

IntroductionAt the prior data cutoff (February 9, 2017) the ALEX trial showed superior investigator-assessed progression-free survival (PFS) for alectinib versus crizotinib in untreated, anaplastic lymphoma kinase (ALK)-positive, advanced NSCLC (hazard ratio = 0.47, 95% confidence interval: 0.34–0.65, p < 0.001). The median PFS in the alectinib arm was not reached versus 11.1 months with crizotinib. Retrospective analyses suggest that the echinoderm microtubule-associated protein-like 4 gene-ALK variant (EML4-ALK) may influence ALK-inhibitor treatment benefit. We present updated analyses, including exploratory subgroup analysis by EML4-ALK variant, after an additional 10 months’ follow-up (cutoff December 1, 2017). MethodsPatients were randomized to receive twice-daily alectinib, 600 mg, or crizotinib, 250 mg, until disease progression, toxicity, death, or withdrawal. PFS was determined by the investigators. Baseline plasma and tissue biomarker samples were analyzed by using hybrid-capture, next-generation sequencing to determine EML4-ALK variant. ResultsBaseline characteristics were balanced. Investigator-assessed PFS was prolonged with alectinib (stratified hazard ratio = 0.43, 95% confidence interval: 0.32–0.58). The median PFS times were 34.8 months with alectinib and 10.9 months with crizotinib. EML4-ALK fusions were detectable in 129 patient plasma samples and 124 tissue samples; variants 1, 2, and 3/ab did not affect PFS, objective response rate, or duration of response. Investigator-assessed PFS was longer for alectinib than for crizotinib across EML4-ALK variants 1, 2, and 3a/b in plasma and tissue. Despite longer treatment duration (27.0 months in the case of alectinib versus 10.8 months in the case of crizotinib), the safety of alectinib compared favorably with that of crizotinib. ConclusionAlectinib continues to demonstrate superior investigator-assessed PFS versus crizotinib in untreated ALK-positive NSCLC, irrespective of EML4-ALK variant.

Comparison of ALK detection by FISH, IHC and NGS to predict benefit from crizotinib in advanced non-small-cell lung cancer

PurposeAnaplastic lymphoma kinase (ALK) is now a validated kinase target in non-small cell lung cancer (NSCLC). We implemented three ALK laboratory methodologies: fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and next-generation sequencing (NGS) to detect EML4-ALK fusions and compared the predictive value for Crizotinib efficacy in ALK-positive patients. Methods55 ALK positive patients confirmed by at least one method were enrolled in the present study, of whom 45 cases were assessed by FISH, IHC and NGS concurrently, and another 10 cases only received IHC and NGS assessment for ALK status. ResultsIHC presented the uppermost positive rate (94.5%), followed by NGS (92.7%) and FISH(82.4%), among which IHC and NGS had the highest concordance rate of 87.3%. No difference was detected in ORR, DCR and PFS of ALK positive cases defined in three groups. Notably, NGS positive patients were correlated with a higher DCR and longer PFS compared to NGS negative cases (P = 0.02 and P = 0.09), while FISH and IHC status were not distinguishing in predicting the outcome of Crizotinib. TP53 concurrent mutation might reduce responsiveness to Crizotinib and worsen prognosis in ALK-rearranged NSCLC. ConclusionFISH present a certain false-negative rate although considered the gold standard. Ventana-D5F3 IHC is qualified as a screening tool, while NGS positive may predict clinical benefit of Crizotinib more accurately, allowing efficient test for specific variants and concurrent genomic alterations.

Discovery Stories of RET Fusions in Lung Cancer: A Mini-Review.

In 2004, a chemical inhibitor of the kinase activity of EGFR was reported to be effective in a subset of lung cancer patients with activating somatic mutations of EGFR. It remained unclear, however, whether kinase fusion genes also play a major role in the pathogenesis of lung cancers. The discovery of the EML4-ALK fusion kinase in 2007 was a breakthrough for this situation, and kinase fusion genes now form a group of relevant targetable oncogenes in lung cancer. In this mini-review article, the discovery of REarrangement during Transfection fusions, the third kinase fusion gene in lung cancer, is briefly described.

A study of ALK-positive pulmonary squamous-cell carcinoma: From diagnostic methodologies to clinical efficacy

BackgroundHigh concordance has been observed between Ventana D5F3 ALK immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) in lung adenocarcinoma (LADC). However, whether a similar conclusion can be applied to lung squamous-cell carcinoma (LSCC) has remained unclear. We therefore evaluated the ALK (anaplastic lymphoma kinase) status and the therapeutic effect of an ALK tyrosine kinase inhibitor (TKI) in IHC- or FISH-positive LSCC. Materials and methodsA total of 2403 LSCC patients from three institutions were screened for ALK aberration by IHC. All IHC-positive cases were subjected to FISH (with an approximately equal number of negative cases as a control group) and next-generation sequencing (NGS). Clinical efficacy was evaluated for the patients who received TKI therapy. ResultsIn 2403 cases of LSCC, 37 cases were identified as ALK-positive by IHC. After quality control, 28 cases were succeeded by FISH (six with insufficient tissue, three with lack of signals) and 13 by NGS (24 failed due to insufficient samples or poor DNA quality); the percentage of non-diagnostic tests was 24.3% (9/37) and 64.9% (24/37), respectively. Four cases (4/2394, 0.17%) analyzed by FISH were determined as ALK-positive. For the control group (40 ALK IHC), FISH demonstrated no samples with ALK gene fusion. The concordance between ALK IHC- and ALK FISH-positive results was 14.3% (4/28). In the 13 cases studied by NGS, two cases showed ALK-EML4 fusion (consistent with two FISH-positive results), and two cases were interpreted as harboring an ALK-association gene mutation. Among four patients (two FISH-positive and two IHC-positive only cases) receiving TKI therapy, two patients had stable disease and the other two had progressive disease. ConclusionsThe positive concordance rate of ALK IHC and FISH in LSCC is far less than that reported for LADC. Therefore, ALK IHC detection in LSCC cannot be used as a diagnostic method for ALK rearrangement.

MiR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

Genome-wide colocalization of RNA-DNA interactions and fusion RNA pairs.

Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA-DNA interactions mapped in two normal, noncancerous cell types [using iMARGI, an enhanced version of the mapping of RNA-genome interactions (MARGI) assay]. Among the top 10 most significant RNA-DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA-DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA-DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA-DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.

Detection of EML4-ALK Fusion Gene Rearrangement in Non-Small Cell Lung Carcinoma in a Sample of Iraqi Patients: A Comparison of Chromogenic in Situ Hybridization with Correlation of ALK Protein Expression

Detection of EML4-ALK Fusion Gene Rearrangement in Non-Small Cell Lung Carcinoma in a Sample of Iraqi Patients: A Comparison of Chromogenic in Situ Hybridization with Correlation of ALK Protein Expression

Non-Small Cell Lung Cancer with Estrogen Receptors and ALK Positivity.

Estrogens are steroid hormones that affect a number of physiological functions in cells; these compounds play an irreplaceable role in the reproductive system. Their effects are mediated by the estrogen receptor (ER), of which there are two subtypes - ER&#945; and ER&#946;. ERs are expressed in regions outside the reproductive system, including bone, brain, intestine, endothelium, kidney, and lung. Expression of ER by lung cancer (LC) cells was first described in the early 1980s. The experimental literature also describes co-expression of ER&#946; and an epidermal growth factor activating mutation, and the additive antiproliferative effects of anti-estrogens plus tyrosine kinase inhibitors during treatment of lung adenocarcinoma. However, coincident expression of ER and ALK translocation were not decribed. The 4-year survival for generalized non-small cell lung cancer indicates the possibility that both molecular features (ER and ALK positivity) may be a favorable prognostic biomarker for this tumor. A 69-year-old patient presented with generalized lung adenocarcinoma that was positive for ERb and the ALK-EML4 fusion gene. The tumor was stage IV. Additional examinations excluded malignancy of the gynecological area. The patient was treated with chemotherapy and atyrosine kinase ALK inhibitor. The role of individual ER subtypes in LC carcinogenesis, and the possible therapeutic effects, is unclear. This is the first documented case of co-occurrence of ALK-EML4 and ER&#946; in LC. Key words non-small cell lung cancer - estrogen receptors - ALK translocation - prognosis - treatment This work was supported by Ministry of Health of the Czech Republic, grant AZV 16-32318A, and by Ministry of Education, Youth and Sports of the Czech Republic, grant NPU I LO1304. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 23. 9. 2018 Accepted: 25. 10. 2018.

P071 Variants Distribution and Heterogeneity of Outcomes to Crizotinib in ALK-Rearranged Chinese Non-Small Cell Lung Cancers

ALK-rearranged NSCLC is a unique molecular subgroup with high sensitivity to ALK inhibitors. Crizotinib, a FDA-approved tyrosine kinase inhibitor for ALK-rearranged NSCLCs, showed remarkable response in ALK-positive NSCLC. However, the magnitude and duration of clinical responses to crizotinib among different ALK variants are found to be heterogeneous, and studies about the clinical outcomes showed contradict conclusions. We collected sequencing information from 110 ALK-positive Chinese NSCLC patients, whose tissue or plasma biopsies were sequenced in a CLIA-certified genomic profiling laboratory. Sequencing results were reviewed with the intent of studying ALK rearrangement distribution and clinical outcomes to crizotinib. A total of 134 (5.6%) ALK rearrangements were identified in a cohort of 1971 NSCLCs, with 39 unique rearrangement partners. EML4 was the most common ALK rearrangement partner, with variant 3 (v3) as the most frequent variants (42.7%) of EML4-ALK fusion, accounting for 71.6% (96/134) of all the rearrangements in 87.3% (96/110) patients. For EML4-ALK positive patients after crizotinib treatment (n=96), survival analysis revealed that patients with EML4-ALK only displayed favorable PFS (10.0 vs 7.2 months, p=0.037) and OS (36.0 vs 20.0 months, p=0.037) than those combined with other fusions. In vitro data reported that variant v3 and v5 was structurally stable and less sensitive to ALK inhibitors due to the lack of TAPE domain. In this study, patients harboring v3 and v5 displayed significantly inferior OS than those with other variants (31 vs 37.6 months, p=0.010). For all the ALK-rearranged patients (n=110), no significant difference was observed between the survival of EML4-ALK and non-EML4-ALK (PFS, 9.4 vs 14.5 months, p=0.61; OS, 35.1 vs 35.5 months, p=0.58), below and above 40-years (PFS, 7.3 vs 11.3 months, p=0.23; OS, 25.4 vs 35.5 months, p=0.69). This study demonstrated the distribution pattern of ALK rearrangements in Chinese NSCLCs, and illustrated the clinical outcomes of ALK-positive patients in different sub-groups. We hope this study could improve basic knowledge of ALK rearrangement and might be helpful for clinicians in choosing patients for appropriate medical treatment. Moreover, these findings advocate for more comprehensive ALK genomic profiling and validation of current results of clinical outcomes in large populations.

A Pilot Prospective Study of Refractory Solid Tumor Patients for NGS-Based Targeted Anticancer Therapy

With the advent of next-generation sequencing (NGS), targeted sequencing is now contributing to decision making for which chemotherapeutics to administer to cancer patients, especially in refractory and metastatic cancer. Given that most patients with refractory cancer develop resistance to chemotherapy and have few treatment options, we performed NGS test to evaluate the efficacy and clinical feasibility of NGS-based targeted anticancer therapy. We used a gene panel for capturing target regions covering 83 cancer-related genes. A total of 25 refractory metastatic solid tumor patients were enrolled in this study. Among the 25 patients, 7 had FDA-approved drug-responsive or -resistant alterations. However, the effectiveness of targeted therapy was assessed by follow-up in three patients (12%). These included crizotinib for ALK-EML4 fusion in a malignancy of undefined origin patient and everolimus for AKT3 amplification in a uterine sarcoma patient. In addition, we identified a KRAS codon 146 mutation (A146V), which is associated with resistance to anti-EGFR, in a cetuximab-resistant colon cancer patient with wild-type KRAS exons 2 and 12, and EGFR amplification. He received bevacizumab therapy. All three patients showed partial response after targeted therapy. Furthermore, we characterized KRAS A146V biologically using colon cancer cells. In conclusion, this study suggests that targeted therapy based on NGS test may be a good choice for improving the care of patients with refractory solid tumors.

Use of Low-Frequency Driver Mutations Detected by Cell-Free Circulating Tumor DNA to Guide Targeted Therapy in Non–Small-Cell Lung Cancer: A Multicenter Case Series

Purpose To evaluate the clinical outcome of patients with non–small-cell lung cancer treated by targeting low variant allelic frequency (VAF) driver mutations identified through cell-free DNA (cfDNA) next-generation sequencing (NGS). Detection of driver mutations in cancer is critically important in the age of targeted therapy, where both tumor-based as well as cfDNA sequencing methods have been used for therapeutic decision making. We hypothesized that VAF should not be predictive of response and that low VAF alterations detected by cfDNA NGS can respond to targeted therapy. Patients and Methods A multicenter retrospective case review was performed to identify patients with non–small-cell lung cancer who received targeted molecular therapy on the basis of findings of low VAF alterations in cfDNA NGS. Mutations at low VAF were defined as &lt; 0.2% mutated cfDNA molecules in a background of wild-type cfDNA. Results One hundred seventy-two patients underwent cfDNA NGS testing. Of the 172 patients, 12 were identified as having low VAF driver alterations and were considered for targeted therapy. The median progression-free survival (PFS) for all patients was 52 weeks (range, 17 to 88 weeks). For patients with EGFR exon 19 deletion (n = 7), the median PFS was 52 weeks (range, 17 to 60.5 weeks). For patients with EML4-ALK fusions (n = 3), the median PFS was 60 weeks (range, 18 to 88 weeks). The median overall survival for all patients after diagnosis was 57.6 weeks. Conclusion Targeted treatment response for driver mutations detected by cfDNA may be independent of VAF, even in relation to other higher VAF aberrations in plasma, and directly dependent on the underlying disease biology and ability to treat the patient with appropriate targeted therapy.

P2.01-78 Validation of InVisionFirst ctDNA NGS Profiling via ddPCR Testing in Patients with Non-Small Cell Lung Cancer (NSCLC)

Tumor tissue based molecular profiling is widely utilized to guide therapy in advanced NSCLC and recently, ctDNA assays have been developed to detect actionable alterations in a non-invasive manner. However, there are frequent reports of discordance between analysis platforms and here we compare the Inivata NGS ctDNA assay with ddPCR based ctDNA analysis and tissue NGS sequencing in patients with advanced NSCLC. InVisionFirst (Inivata) is a ctDNA NGS assay for detection of genomic alterations in 36 genes commonly mutated in NSCLC and other cancer types. A cohort of 52 patients underwent ctDNA analysis by InVisionFirst and were tested in a blinded manner for 4 actionable gene alterations (EGFR L858R and Ex19del, KRAS G12C and G12D) and 30 of the cohort also had testing for 5 additional alterations (EGFR T790, EML4-ALK fusions, ROS1 fusions, KRAS G12V and BRAF V600E) at one of two independent ddPCR laboratories. Finally, tissue analysis by NGS was available in 21 patients to arbitrate any discordance between the results of the 2 liquid biopsy techniques. Across the 9 specific genetic alterations investigated by both ctDNA platforms, 26 alterations were detected by the InvisionFirst platform and 23 were detected by the ddPCR platforms. The overall concordance of gene alterations was 98.5% (320/342) with positive agreement of 95.5% and negative agreement of 98.8%. Discordance was observed in 6 detected gene alterations, with 1 EML4-ALK fusion, 1 EGFR exon19 deletion and 2 KRAS G12Cs being detected by InVisionFirst but not by ddPCR, and 1 EGFR L858R and 1 KRAS G12D detected by ddPCR but not InVisionFirst. One G12C, the EML4-ALK, and the L858R alteration were confirmed by tissue NGS. The KRAS G12D was not confirmed by tissue NGS. No tissue was available to examine for the detection of the EGFR exon 19 deletion or the other KRAS G12C mutation. All other results were concordant. This study in NSCLC patients demonstrates excellent concordance of the InVisionFirst ctDNA NGS assay with ddPCR based ctDNA analysis via blinded independent laboratories. The excellent sensitivity and specificity support the use of the InVisionFirst assay as a non-invasive “liquid biopsy” for molecular profiling.

P2.09-10 qRT-PCR as an Efficient and Reliable Diagnostic Screening Approach for the Detection of EML4-ALK Fusion Gene in NSCLC Samples: A Pilot Study

P2.09-10 qRT-PCR as an Efficient and Reliable Diagnostic Screening Approach for the Detection of EML4-ALK Fusion Gene in NSCLC Samples: A Pilot Study

Desmoplastic Infantile Ganglioglioma/Astrocytoma (DIG/DIA) Are Distinct Entities with Frequent BRAFV600 Mutations.

Desmoplastic infantile ganglioglioma (DIG) and desmoplastic infantile astrocytoma (DIA) are extremely rare tumors that typically arise in infancy; however, these entities have not been well characterized in terms of genetic alterations or clinical outcomes. Here, through a multi-institutional collaboration, the largest cohort of DIG/DIA to date is examined using advanced laboratory and data processing techniques. Targeted DNA exome sequencing and DNA methylation profiling were performed on tumor specimens obtained from different patients (n = 8) diagnosed histologically as DIG/DIGA. Two of these cases clustered with other tumor entities, and were excluded from analysis. The remaining 16 cases were confirmed to be DIG/DIA by histology and by DNA methylation profiling. Somatic BRAF gene mutations were discovered in 7 instances (43.8%); 4 were BRAFV600E mutations, and 3 were BRAFV600D mutations. Three instances of malignant transformation were found, and sequencing of the recurrence demonstrated a new TP53 mutation in one case, new ATRX deletion in one case, and in the third case, the original tumor harbored an EML4-ALK fusion, also present at recurrence. DIG/DIA are distinct pathologic entities that frequently harbor BRAFV600 mutations. Complete surgical resection is the ideal treatment, and overall prognosis is excellent. While, the small sample size and incomplete surgical records limit a definitive conclusion about the risk of tumor recurrence, the risk appears quite low. In rare cases with wild-type BRAF, malignant progression can be observed, frequently with the acquisition of other genetic alterations.Implications: DIG/DIA are a distinct molecular entity, with a subset frequently harboring either BRAF V600E or BRAF V600D mutations. Mol Cancer Res; 16(10); 1491-8. ©2018 AACR.

136P - EML4-ALK fusion variants associate with gender and age in Chinese NSCLC patients

136P - EML4-ALK fusion variants associate with gender and age in Chinese NSCLC patients

ES04.03 Overcoming Resistance by Improving Treatment Compliance

ES04.03 Overcoming Resistance by Improving Treatment Compliance

Parp3 promotes long-range end joining in murine cells

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

P15 Prevalence of EML4- ALK Fusion Gene in Adenocarcinoma Lung Patients by Using Immuno Histo Chemistry

An oral ATP- competitive TKI of ALK and c- MET, Crizotinib has shown impressive clinical activity in advanced non–small cell lung cancers especially in the tumors harboring ALK rearrangements. With FISH as the mainstay for detection of ALK rearrangements, the ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, IL) has become an FDA-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The objective of this molecular epidemiological study is to estimate the prevalence of EML4-ALK fusion gene using IHC as a cost effective alternative to FISH for Indian patients with adenocarcinoma lung. Patients with NSCLC, adenocarcinoma histology, whose tumors had been tested for EML4-ALK fusion gene using IHC were considered for this study. Permission was obtained from the Ethics committee before the start of the study. Clinical characteristics and treatment details were collected from the patient’s medical records. IHC analysis was performed using a Ventana automated immunostainer (Ventana Medical Systems, Illkirch Graffenstaden, France). Detection was performed using a multimer-technology system with the UltraView Universal DAB detection kit. A total of 204 NSCLC adenocarcinoma patients were included in the study. There were 126 (61.7%) men and 78 (38.23%) women with a median age of 57 years. Of the 204 patients, 47 (23.03%) were non-smokers and 175 (85.78%) had stage-IV disease at the time of initial diagnosis. 48 (23.52%) blocks were positive for EGFR mutations whereas 156 (76.47%) were EGFR wild type. EML4-ALK fusion gene was present in 27 (13.23%) patients whereas 177 (86.76%) tumors were EML4-ALK negative. 25 out of the 27 patients with ALK positivity received Crizotinib therapy. The incidence of EML4-ALK gene fusions (13.23%) in this Indian population is four fold high than the previous reported incidences and supports the claim of several recent studies that a relatively new ALK clone, 5A4 and D5F3 from cell signaling technology (Ventana) can accurately identify ALK rearranged lung ACA as compared to FISH. The inclusion of IHC for the detection of EML4-ALK gene fusions as a low cost alternative seems warranted.