EML4-ALK Translocation Research Articles

Prolonged disease control despite ALK inhibitor discontinuation in advanced ALK-positive NSCLC

Introduction: EML4-ALK is an oncogenic driver, seen in around five per cent of advanced non-small-cell lung cancer (NSCLC) patients, which can be targeted with anaplastic lymphoma kinase tyrosine kinase inhibitors with great response rates. Disease flare refers to sudden rapid disease worsening on tyrosine kinase inhibitors (TKI) discontinuation, which is associated with shorter survival and worse outcomes. Here, we review cases previously published in the literature where patients developed disease flares, and contrast this with our patients who had prolonged survival despite TKI discontinuation. Case description: We report three different patients with advanced ALK-positive NSCLC seen at our institute, who had EML4-ALK translocation variant 1 oncogenic driver on next-generation sequencing. They received treatment with several different ALK inhibitors before opting to discontinue TKI. They were able to come off TKI safely without developing disease flare and had prolonged survival. Discussion: Shorter time to progression on TKI, presence of symptoms with disease progression or central nervous system/pleural metastasis have been previously linked with development of flare, although this was not seen in our case series. Tumour response at the time of treatment discontinuation, line of therapy, overall disease burden, fusion variant and co-alteration status can affect the prognosis of these patients after ALK TKI cessation. In particular, variant 1 and wild-type TP53 status may be a suitable patient population for dose optimisation strategies. Intermittent TKI dosing strategies may help to avoid acquiring resistance mutations and prevent long-term treatment toxicities. Conclusion: It is important for clinicians to identify patients at risk for developing disease flare on TKI discontinuation to improve outcomes. Intermittent TKI dosing strategies require further investigation.

Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy.

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma.

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary). Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit. ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation. ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

The Change in Paradigm for NSCLC Patients with EML4-ALK Translocation.

The severe prognosis linked with a lung cancer diagnosis has changed with the discovery of oncogenic molecularly driven subgroups and the use of tailored treatment. ALK-translocated advanced lung cancer is the most interesting model, having achieved the longest overall survival. Here, we report the most important paradigmatic shifts in the prognosis and treatment for this subgroup population occurred among lung cancer.

State-of-the-Art Molecular Oncology of Lung Cancer in Taiwan.

Lung cancers are life-threatening malignancies that cause great healthcare burdens in Taiwan and worldwide. The 5-year survival rate for Taiwanese patients with lung cancer is approximately 29%, an unsatisfactorily low number that remains to be improved. We first reviewed the molecular epidemiology derived from a deep proteogenomic resource in Taiwan. The nuclear factor erythroid 2-related factor 2 (NRF2)antioxidant mechanism was discovered to mediate the oncogenesis and tumor progression of lung adenocarcinoma. Additionally, DNA replication, glycolysis and stress response are positively associated with tumor stages, while cell-to-cell communication, signaling, integrin, G protein coupled receptors, ion channels and adaptive immunity are negatively associated with tumor stages. Three patient subgroups were discovered based on the clustering analysis of protein abundance in tumors. The first subgroup is associated with more advanced cancer stages and visceral pleural invasion, as well as higher mutation burdens. The second subgroup is associated with EGFR L858R mutations. The third subgroup is associated with PI3K/AKT pathways and cell cycles. Both EGFR and PI3K/AKT signaling pathways have been shown to induce NRF2 activation and tumor cell proliferation. We also reviewed the clinical evidence of patient outcomes in Taiwan given various approved targeted therapies, such as EGFR-tyrosine kinase inhibitors and anaplastic lymphoma kinase (ALK)inhibitors, in accordance with the patients’ characteristics. Somatic mutations occurred in EGFR, KRAS, HER2 and BRAF genes, and these mutations have been detected in 55.7%, 5.2%, 2.0% and 0.7% patients, respectively. The EGFR mutation is the most prevalent targetable mutation in Taiwan. EML4-ALK translocations have been found in 9.8% of patients with wild-type EGFR. The molecular profiling of advanced NSCLC is critical to optimal therapeutic decision-making. The patient characteristics, such as mutation profiles, protein expression profiles, drug-resistance profiles, molecular oncogenic mechanisms and patient subgroup systems together offer new strategies for personalized treatments and patient care.

Abstract A026: Altered immune landscape in aging lungs contributes to malignant evolution

Abstract Aging is a major risk factor in increased lung cancer incidence. While most research has focused on age-associated mutation accumulation to explain the late-life increase in cancer incidence, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. Chronic, low-level inflammation has been associated with aging, termed inflammaging, yet how age-associated changes in lung tissue microenvironments contribute to increased lung cancer incidence has remained largely unknown. Since chronic inflammation has been shown to contribute to tumor development, we hypothesized that inflammaging contributes to increased oncogenic adaptation in the lung. Using either viral delivery of CRISPR constructs to mediate EML4-ALK translocations or ectopic expression of KRAS-G12D, we showed increased adenoma formation in old mice. Importantly, in the EML4-ALK model, we showed that the overexpression of alpha-1 antitrypsin (AAT) in old mice resulted in lower adenoma counts compared to their old wild type counterparts. Flow cytometric analysis of immune cells isolated from bronchoalveolar fluid of young and old mice showed an altered immune landscape, such as increased neutrophils, gamma delta T cells, and Foxp3+ regulatory T cells. Furthermore, analysis of the single-cell RNAseq data from Tabula Muris Consortium demonstrated increased exhaustion markers in the CD8+ T cells and regulatory T cells. Separately, Gene Set Enrichment Analysis (GSEA) of the differential gene expressions of lung epithelial cells isolated from young and old mice revealed enriched pathways related to immune activation and inflammatory response, and immune-suppression markers. Lastly, bulk RNA-seq from lungs of young, old, and old mice overexpressing AAT revealed increased immune cell exhaustion markers and that the overexpression of AAT partially reversed this increase. Finally, analysis of Genotype-Tissue Expression (GTEx) data comparing gene expressions in lungs of young and old humans similarly showed enriched pathways related to immune activation and increased T cell exhaustion markers in the elderly. In addition, using deconvolution methods CiberSort and xCell, we demonstrated altered innate and adaptive immune cell populations, for example, increased neutrophils and regulatory T cells, that are associated with advanced age, similar to aging mice. In conclusion, we showed that there is an exhausted immune microenvironment in aging lungs, that inflammation contributes to the increased tumor initiation, and that decreasing inflammation could decrease the lung tumor incidence by reactivating the immune system. Citation Format: Shi Biao Chia, Catherine Pham-Danis, Hannah Scarborough, Nathaniel Little, Etienne P. Danis, Andrew E. Goodspeed, Charles Dinarello, James DeGregori. Altered immune landscape in aging lungs contributes to malignant evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A026.

WBRT for brain metastases from non-small cell lung cancer: for whom and when?-Contemporary point of view.

The incidence of brain metastases (BM) is estimated between 20% and 40% of patients with solid cancer. The most common cause of this failure is lung cancer, and in locally advanced non-small cell lung cancer (NSCLC) BM represent a common site of relapse in 30–55% cases. The basic criteria of therapeutic decision-making are based on the significant prognostic factors which are components of prognostic scores. The standard approach to treatment of BM from NSCLC include whole brain radiotherapy (WBRT) which is used as adjuvant modality after local therapy (surgery or stereotactic radiosurgery) or as primary treatment and it remains the primary modality of treatment for patients with multiple metastases. WBRT is also used in combination with systemic therapy. The aim of presented review of literature is trying to answer which patients with BM from NSCLC should receive WBRT and when it could be omitted. There were presented the aspects of application of WBRT in relation to (I) choice between WBRT or the best supportive care and (II) employment of WBRT in combination with local treatment modalities [surgical resection or stereotactic radio-surgery (SRS)] and/or with systemic therapy. According to data from literature we concluded that the most important factor that needs to be considered when assessing the suitability of a patient for WBRT is the patient’s prognosis based on the Lung-molGPA score. WBRT should be applied in treatment of multiple BM from lung cancer in patients with favourable prognosis and in in patients with presence of EML4-ALK translocation before therapy with crizotinib. Whereas WBRT could be omitted in patients with poor prognosis and after primary SRS.

Relationship between clinical features and gene mutations in non-small cell lung cancer with osteoblastic bone metastasis

ObjectiveLung cancer patients presenting with osteoblastic bone metastases at the first visit is rare. We investigated the clinical characteristics and gene mutation rate of non-small cell lung cancer patients with osteoblastic bone metastases at the time of the initial diagnosis. Materials and MethodsWe retrospectively screened newly diagnosed non-small cell lung cancer patients with osteoblastic bone metastases who presented from June 2015 to March 2021, and analyzed their clinical characteristics and status of EGFR gene mutations, EML4-ALK translocation and ROS1 rearrangements. For comparison, we collected data from patients with non-small cell lung cancer who had osteolytic bone metastases at their first visit between June 2015 and March 2021. ResultsFifty patients had bone metastases at the initial diagnosis. Among them, eight patients (8/50 = 16%) had osteoblastic bone metastases, and the lung tumors in all of them were histopathologically adenocarcinomas. Among the eight cases, two were EGFR mutation-positive, none were EML4-ALK translocation-positive, two were ROS1 rearrangement-positive, and the remaining four cases were negative for all three gene mutations/rearrangements. Compared with the osteolytic bone metastasis group, the percentage of non-smokers was higher (p = 0.020) and the ROS1 rearrangement positivity rate was higher (p = 0.05) in the osteoblastic bone metastasis group. ConclusionOur results indicate that osteoblastic bone metastases in NSCLC are suggestive of adenocarcinoma, and that a high proportion of these patients might be positive for ROS1 rearrangements, and hence, indicated for more aggressive diagnostic biopsies.

Abstract 2400: Strong biological bias for ALK intron 19 breakpoints in NSCLC

Abstract Introduction: EML4-ALK translocations are detected in 4-8 % of non-small cell lung cancer (NSCLC). While different EML4-ALK variants are defined by different breakpoints in the EML4 gene, most frequently located in intron 6 or 13, ALK breakpoint is almost invariably in intron 19. Rare reports describe EML4-ALK translocations with breakpoints in intron 17 or intron 18 of the ALK gene. Despite all these ALK breakpoints potentially generate oncogenic fusions, the reasons of this strong imbalance toward intron 19 (exon 20) breakpoints in ALK positive NSCLC are currently unknown. The aim of this study is to investigate the mechanisms that underlie ALK translocation in NSCLC. Methods: Eight different EML4-ALK fusions were either overexpressed with a retroviral system or generated with CRISPR/Cas9 lentivirus from the endogenous loci. EML4 exons 1-6 (E6) or exons 1-13 (E13) with different ALK exons: E6;A18, E6;A19, E6;A20 or E6;A21 and E13;A18, E13;A19, E13;A20, E13;A21. The fusions E6;A21 and E13;A21 contained an early stop codon not producing functional proteins. These EML4-ALK fusions were expressed in NIH3T3 and PC9 by retroviral vectors or engineered in PC9 (an EGFR-dependent lung cancer cell line that is sensitive to inhibition with the EGFR inhibitor osimertinib) and BEAS-2B cells by CRISPR/Cas9. HTGTS was performed on engineered PC9 cells. NSG immunodeficient mice were used for in vivo experiments with NIH3T3 and BEAS-2B cells. Results: The retroviral overexpression system showed that all EML4-ALK fusion proteins were expressed leading to an actively phosphorylated ALK in NIH3T3 and PC9 cells, except E6;A21 and E13;A21 fusions where no protein was detected, as expected. Accordingly, all active variants were able to induce NIH3T3 cell transformation and tumor formation in vivo and conferred resistance to osimertinib in PC9 cells. In contrast, when EML4-ALK translocations were induced by CRISPR/Cas9 in PC9 cells, only E6;A20 and E13;A20 variants rapidly overcame osimertinib inhibition while other variants rescued osimertinib-inihibited cells slowly and less efficiently. Surprisingly, the E6;A20 and E13;A20 fusions were invariably found in all resistant clones independently of the translocation induced by CRISPR/Cas9. Similarly, all tumors formed in vivo by BEAS-2B cells contained the E6;A20 or E13;A20 variants irrespective of the translocation induced originally. By cloning translocation junctions at large scale with HTGTS, we found that clones initially forced to break into ALK intron 17 eventually acquired a second breakpoint in ALK intron 19. Conclusions: Our data show that all EML4-ALK fusion variants were equally oncogenic when overexpressed. In contrast, when EML4-ALK variants were generated from the endogenous loci there was a strong selection bias toward ALK fusions originating in intron 19 suggesting that intron 19 variants have the strongest oncogenic potential in lung epithelial cells. Citation Format: Giulia C. Leonardi, Taek-Chin Cheong, Chiara Ambrogio, Tao Chen, Wei-Tien Tai, Elif Karaca, Ines Mota, Massimo Libra, Mark M. Awad, Rafael Blasco-Patino, Roberto Chiarle. Strong biological bias for ALK intron 19 breakpoints in NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2400.

Abstract 184: SureSelect sequencing panels and algorithms to detect copy number variations (CNVs), DNA rearrangements, microsatellite Instability and tumor mutational burden (TMB) in FFPE specimens

Abstract Background: The current standard of genomic profiling of cancer tissues relies upon multiple separate technologies to aid in tumor characterization. Here we describe an NGS panel and analysis workflow for cancer samples that can detect single nucleotide polymorphisms (SNPs), insertions and deletions (INDEL), somatic copy number alterations (SCNAs), and translocations (TLs), Tumor Mutation Burden (TMB) and Microsatellite Instability (MSI) in a single assay. Methods: We have developed a SureSelect target enrichment sequencing panels for comprehensive profiling of single nucleotide variations (SNP/INDEL), gene copy number variations (CNV), and DNA translocations (TL) and relevant microsatellite regions in a single assay. The TMB panel gene list was curated based on genes found in cancer gene databases such as Clinical Interpretations of Variants in Cancer (CIViC), Cancer Genome Interpreter (CGIdb), Catalog of Somatic Mutations in Cancer Census (COSMIC), Precision Oncology Knowledge Base (OncoKB). An accompanying data analysis pipeline will provide highly sensitive and accurate detection of variants and allows for the determination TMB and MSI status. Probes have been selected for over 500 genes to detect SCNAs and translocation in several cancer subtypes. The probes are optimized with the SureSelect XTHS protocol, which enables sensitive and accurate detection of rare mutation events within a heterogeneous sample with molecular barcode-mediated error correction. The SCNAs probes target regions in the genome that help in decreasing the noise for SCNAs calling. The data analysis utilizes either a matched normal or standard control DNA to compute read depth log ratios. Aberrant regions of constant ploidy are first identified followed by the application of a variational streaming algorithm on log ratio and SNP allele frequencies to determine the major clones present in the tumor. Translocation detection was verified on lung cancer samples by analyzing split reads across the fusion breakpoints. Results: By diluting samples with known copy-number aberrations into normal DNA, a limit of detection of ⇐2.3 copies/cell was demonstrated, equivalent to the detection of 3 copies at 30% tumor fraction. A similar approach demonstrated an ability to detect EML4-ALK and SLC24A2-ROS1 translocations at 3% allele frequency. Performance of the assay was further evaluated using 100+ FFPE samples harboring rearrangements, gene amplifications, and SNP/Indels. Concordance to the reference methods (FISH, WES, WGS) was >90%. TMB and MSI algorithms were benchmarked using in silico analysis of TCGA data and performance of the assay was evaluated on FFPE samples with orthogonally-determined MSI/TMB status. Conclusions: This work represents an important advancement in the development of a single assay to detect copy number variation, DNA rearrangement and mutations, TMB and MSI status of a FFPE sample. Citation Format: Arjun Vadapalli, Akanksha Khare, Hanjun Shin, Ashutosh Ashutosh, Linus Forsmark, Anne Lucas, Scott Happe, Gilbert Amparo, Carlos Pabon, Jayati Ghosh, Tracy Liu, Jimmy Jin, Mike Ruvolo, Douglas Roberts. SureSelect sequencing panels and algorithms to detect copy number variations (CNVs), DNA rearrangements, microsatellite Instability and tumor mutational burden (TMB) in FFPE specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 184.

Clinicopathological and Prognostic Significance of the EML4-ALK Translocation and IGFR1, TTF1, Napsin A Expression in Patients with Lung Adenocarcinoma.

Patients with lung adenocarcinoma who harbor ALK gene rearrangements can demonstrate significant clinical benefit with ALK tyrosine kinase inhibitors. Insulin-like growth factor receptor 1 (IGFR1) is a cellular membrane receptor that is overexpressed in many tumors. It plays an important role in cancer progression and is associated with increased postoperative recurrence and poorer disease-free survival. The aim of this study was to determine the EML4-ALK mutation and IGFR1 expression in lung adenocarcinoma and analyze their prognostic value. In this study, we analyzed the EML4-ALK mutation using the FISH and IHC techniques in 251 lung adenocarcinoma (203 primary resections, 48 metastasectomies) cases. Correlative analyses were performed between the EML4-ALK mutation, the IGFR1, TTF1, and NapsinA expression, and the clinicopathologic factors in lung adenocarcinomas. The EML4-ALK mutation was observed in 3.8% of the cases and it was associated with the solid pattern, signet ring cell morphology, and larger tumor size. IGFR1 expression was identified in 49% of the cases and most of the ALK-mutated cases were also expressing the IGFR1 protein (66%). IGFR1 expression frequency was increased in metastasectomy specimens. A solid signet-ring cell pattern or mucinous cribriform pattern was present at least focally in all ALK-positive tumors, consistently with the literature. In addition, IGFR1 expression levels showed an increase in the EML4-ALK-mutated cases in our series, but the clinical significance of this finding should be supported by larger series and survival analysis. Our findings show that IGFR1 expression may be useful as a poor prognostic marker in patients with lung adenocarcinoma.

EP1.03-32 Prevalence of EGFR and ALK Mutations in Non Small Cell Lung Cancer in Viet Nam National Cancer Hospital

A significant percentage of non small cell lung cancer (NSCLC) have driver mutations. Epidermal growth factor receptor (EGFR) mutations and anaplastic large-cell lymphoma kinase (ALK) rearrangements are now routine biomarkers that have been incorporated into the practice of managing NSCLC. To date, there has been no assessment of the prevalence of these mutations in NSCLC in Viet Nam National Cancer Hospital. The prospective study of formalin fixed paraffin embedded (FFPE) tissues from patients diagnosed with NSCLC was performed to assess the prevalence of EGFR and ALK mutations in NSCLC in Viet Nam National Cancer Hospital. Patients with NSCLC in Viet Nam National Cancer Hospital were prospectively enrolled. FFPE tissue samples were tested for EGFR mutation by PCR and for EML4-ALK translocation by fluorescence in situ hybridization (FISH) A total of 103 patients were enrolled, 78 (76.7%) males and 25 females (23.3%), with a mean age of 57.7 years. Adenocarcinoma and Squamous cell carcinoma rate were 89.3% and 10.7%, respectively. EGFR testing of 103 patients (100%) demonstrated the wild type in 76 (73.8%) and mutated forms in 27 (26.2%). Some 74.0% of EGFR positive patients were non-smokers and 44.4% were females. Regarding the EML4-ALK translocation, testing in 103 cases (100%) gave positive results in only 11 (10.7%). Among 11 patients with EML4-ALK translocation, 6 patients (54.5 %) were females and 6 patients (54.5 %) were smokers. Moreover, only two squamous cell carcinoma patients had positive EGFR mutation and only one patient had concurrent EGFR mutation and ALK rearrangement In this sudy investigating the prevalence of EGFR and ALK mutations in non small cell lung cancer in Vietnam National Cancer Hospital, 26.2% had EGFR mutation and 10.7% had ALK translocation mutations, as compared to 35% and 6.1%, respectively, in Asian

Dual drive coexistence of ALK rearrangement and KRAS mutation advanced lung adenocarcinoma and response to crizotinib

Chromosomal translocation resulting in the fusion between the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene has been considered as a novel oncogenic fusion in a subset of non-small cell lung cancer (NSCLC), mostly in non-smokers with adenocarcinoma. EML4-ALK translocations are commonly reported to be mutually exclusive with epidermal growth factor receptor (EGFR) or KRAS mutations. Herein, we reported a rare case of 47-year-old female was diagnosed with lung adenocarcinoma and treated with three cycles of chemotherapy. A biopsy acquired after disease progression revealed concurrent KRAS mutation and ALK translocation by an next-generation sequencing (NGS) assay. The patient had a favorable tumor response to crizotinib, a tyrosine kinase inhibitor (TKI). A further understanding of the molecular biology with multiple oncogenic drivers will promote the optimal treatment for NSCLC.

Dual drive coexistence of ALK rearrangement and KRAS mutation advanced lung adenocarcinoma and response to crizotinib

Chromosomal translocation resulting in the fusion between the echinoderm microtubule-associated protein-like 4 ( EML4 ) gene and the anaplastic lymphoma kinase ( ALK ) gene has been considered as a novel oncogenic fusion in a subset of non-small cell lung cancer ( NSCLC ), mostly in non-smokers with adenocarcinoma. EML4-ALK translocations are commonly reported to be mutually exclusive with epidermal growth factor receptor (EGFR) or KRAS mutations. Herein, we reported a rare case of 47-year-old female was diagnosed with lung adenocarcinoma and treated with three cycles of chemotherapy. A biopsy acquired after disease progression revealed concurrent KRAS mutation and ALK translocation by an next-generation sequencing (NGS) assay. The patient had a favorable tumor response to crizotinib, a tyrosine kinase inhibitor (TKI). A further understanding of the molecular biology with multiple oncogenic drivers will promote the optimal treatment for NSCLC.

Frequency of actionable alterations in epidermal growth factor receptor (EGFR) wild type non-small cell lung cancer: experience of the Wide Catchment Area of Romagna (AVR).

Molecular diagnostics for non-small cell lung cancer (NSCLC) has become the standard of care for personalized treatment. Epidermal growth factor receptor (EGFR) mutation and EML4-ALK translocation represent the two most important alterations in first-line treatment decision-making. However, other potentially targetable alterations are also present. One thousand consecutive NSCLC patients with EGFR wild type (wt) tumors diagnosed by routine molecular analysis were considered. KRAS, BRAF, ERBB2, PIK3CA, NRAS, ALK, MAP2K1, RET and DDR2 gene mutations were analyzed using the multiparametric Sequenom MassARRAY® platform. EML4-ALK and ROS1 rearrangements were also assessed by fluorescent in situ hybridization. HER4 status was determined by direct sequencing. Three hundred and forty-eight (34.8%), 31 (3.1%), 39 (4.4%), 14 (1.8%), 6 (0.7%), 16 (1.8%), 5 (0.6%) and 9 (0.9%) patients showed an alteration in KRAS, BRAF, ALK, ROS1, NRAS, PIK3CA, MAPK1/2 and HER2 genes, respectively. Of the 657 patients for whom all markers were determined, 318 (48%) patients had at least one alteration. Eight patients showed overlapping mutations, 4 KRAS mutation/EML4-ALK translocation, one KRAS mutation/ROS1 rearrangement, 2 KRAS/PIK3CA mutations, and one BRAF/PIK3CA mutations. About 50% of our patients had a potentially targetable alteration, confirming the usefulness of a multiparametric approach for routine molecular diagnostics aimed at identifying potential therapeutic targets.

Abstract 1099: Molecular drivers of neutrophil recruitment to primary non-small cell lung cancer

Abstract Neutrophils are associated with developing cancer lesions and are the main immune component of primary non-small cell lung cancer (NSCLC). Multiple studies support the notion that tumor associated neutrophils (TANs) can promote tumor progression. We hypothesize that there is a hierarchy of molecular cues produced by developing lung cancers that guide circulating neutrophils to infiltrate the tumor microenvironment and become TANs. Identifying these cues may permit modulation of neutrophil infiltration within developing lung cancers and may thereby act as an immunotherapeutic tool to suppress cancer progression and improve response to existing therapeutics. To this end, we profiled 5 established NSCLC cell lines representing the common NSCLC subtypes using a qRT-PCR 84 gene panel (A549; KRAS mutant, PC9; EGFR mutant, H1993; MET amplification, H3122; EML4-ALK translocation, HCC78; ROS1 translocation). We focused on 4 of the most commonly upregulated genes in all cell lines, which were osteopontin (Spp1), vascular endothelial growth factor A (VEGF-A), macrophage inhibitory factor (MIF), and C-X-C motif ligand 1 (CXCL1). After confirming protein expression of these targets by western blot, we performed shRNA knock down (KD) of these genes and tested the migration of neutrophils towards treated and control cell lines in a novel microfluidic device that allows increased throughput studies of neutrophil attractants. Findings from KD experiments were confirmed via antibody-mediated inhibition. We observed a 3-fold increase of neutrophil migration to the A549 cancer cell line compared to the serum free control (p=0.0265). Furthermore, this increase was inhibited in Spp1 (64% decrease), MIF (84%), VEGF (82%) KDs and their corresponding neutralizing antibodies. We have therefore identified 4 proteins that play a key role in neutrophil recruitment to NSCLC cell lines in vitro and have demonstrated the application of a simple microfluidic device to test neutrophil migration patterns. This data provides the basis for in vivo investigations to elucidate the key molecular cues for neutrophil infiltration within developing lung cancers. Citation Format: Claire Wang, Roni Rayes, Jack Mouhanna, Betty Giannias, Arvind Chandrasekaran, Rachel Mot, Christopher Moraes, Sidong Huang, Jonathan Cools-Lartigue, Nicholas Bertos, Lorenzo Ferri, Jonathan Spicer. Molecular drivers of neutrophil recruitment to primary non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1099.

Oncogene addiction and radiation oncology: effect of radiotherapy with photons and carbon ions in ALK-EML4 translocated NSCLC

BackgroundPatients with Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) positive lung cancer are sensitive to ALK-kinase inhibitors. TAE684 is a potent second generation ALK inhibitor that overcomes Crizotinib resistance. Radiotherapy is an integral therapeutic component of locally advanced lung cancer. Therefore, we sought to investigate the effects of combined radiotherapy and ALK-inhibition via TAE684 in ALK-positive vs. wild type lung cancer cells.MethodsHuman non-small cell lung cancer (NSCLC) cell lines harboring wild-type ALK (A549), EML4-ALK translocation (H3122) and murine Lewis Lung Cancer (LLC) cells were investigated. Cells were irradiated with 1–4 Gy X-Rays (320 keV) and carbon ions (Spread-out Bragg Peak, SOBP (245.4–257.0 MeV/u)) at Heidelberg Ion Therapy center. TAE684 was administered at the dose range 0–100 nM. Clonogenic survival, proliferation and apoptosis via caspase 3/7 expression level were assessed in all three cell lines using time-lapse live microscopy.ResultsTAE684 inhibited the proliferation of H3122 cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of ~ 8.2 nM. However, A549 and LLC cells were relatively resistant to TAE684 and IC50 was not reached at concentrations tested (up to 100 nM) in proliferation assay. The antiproliferative effect of TAE684 was augmented by radiotherapy in H3122 cells. TAE684 significantly sensitized H3122 cells to particle therapy with carbon ions (sensitizer enhancement ratio ~1.61, p < 0.05). Caspase 3/7 activity was evidently enhanced after combination therapy in H3122 cells.ConclusionsThis is the first report demonstrating synergistic effects of combined TAE684 and radiotherapy in EML4-ALK positive lung cancer cells. In addition to conventional photon radiotherapy, ALK-inhibition also enhanced the effects of particle irradiation using carbon ions. Our data indicate beneficial effects of combined ALK-inhibition and radiotherapy in treatment of this distinct subpopulation of NSCLC that warrant further evaluation.

An international epidemiological analysis of young patients with non-small cell lung cancer (AduJov-CLICaP)

BackgroundA proportion of patients with NSCLC is diagnosed at 40 years or younger. These patients tend to be never-smokers, usually present with stage IV adenocarcinoma, and have somatic genomic alterations. Few studies have documented and analyzed epidemiological characteristics of this population. Materials and methodsWe performed an international epidemiological analysis of 389 young patients with NSCLC. Data was collected from centers participating in the Latin American Consortium for Lung Cancer Research (AduJov-CLICaP). Patients were identified and data was retrospectively collected from different Latin American countries and Canada (Argentina=6, Canada=19, Colombia=29, Costa Rica=9, Mexico=219, Nicaragua=2, Panama=19, Perú=76 and Venezuela=10). The period of study was from 2012 to 2017. Inclusion criteria were: age 40 years or less and a histologically confirmed NSCLC. Clinical data was obtained, and EGFR mutation status and EML4-ALK translocation were collected. ResultsNSCLC patients aged 40 years or less accounted for approximately 4% of the total NSCLC population. Female patients accounted for 54.5%, while median age was of 37 years. Adenocarcinoma accounted for 86.1% (n=335/389), 72.5% (n=282/389; unknown=5) of patients were non-smokers, and 90.3% (n=351/389) had stage IV disease. Site of metastasis was obtained from 260/351 (unknown=91) stage IV patients (lung metastasis=40.0%, CNS metastasis=35.7%, and bone metastasis=31.5%). OS for the total population was 17.3 months (95%CI=13.9-20.7). OS for EGFRm(+)=31.4months (95%CI=11.6-51.3), EGFRm(−)=14.5months (95%CI=11.0–17.9) (p=0.005). OS for alk(+)=9.8months (95%CI=3.1-16.5) and alk(−)=5.6months (95%CI=3.9–7.3) (p=0.315). ConclusionsPatients aged 40 years or less account for a small but important proportion of NSCLC cases. Younger patients may have different characteristics compared to the older population. EGFRm and EML4-alk translocation frequency is higher than that of the general population.

Abstract 1737: ALK rearrangement analysis in circulating tumor cells of lung cancer patients

Abstract The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information. Citation Format: Min Kyung Jeon, Young Hun Kim, Eunjoo Hwang, Hye Seon Lee, Ji-hyun Uh, Myoung Shin Kim, JooKyung Park, Byung Hee Jeon, Se-Hoon Lee. ALK rearrangement analysis in circulating tumor cells of lung cancer patients [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 1737. doi:10.1158/1538-7445.AM2017-1737

Abstract 2207: HSP90 inhibition leads to a differential proteomics profile in non small cell lung cancer cell lines

Abstract Background: The cancer chaperone, Heat Shock Protein 90 (HSP90), is known for its ability to regulate the stability of different oncogenic proteins. Thus, its overexpression has been related to unfavorable prognosis in some types of tumors. EGFR and EML4-ALK, two of the most important drivers in non-small cell lung cancer (NSCLC), are HSP90 clients and extremely depend on it. As a consequence, this chaperone is especially important in NSCLC hence HSP90 inhibition shows a lot of possibilities to future treatments in this tumor type. Nevertheless, to obtain a successful clinical development, will be essential supporting evidence of inhibitory efficacy in several molecularly defined subgroups of NSCLC. Methods: NSCLC cell lines carrying different gene mutations, whose direct (HCC827: EGFR mutated and H3122: EML4-ALK rearrangement) and indirect (A549: KRAS mutated) relationship with HSP90 has been reported, were used. In these cell lines, along with H1781 (EGFR, KRAS, ALK wild type) as control, the activity of the chaperone studied was interrupted. To this end, pharmacological inhibition of HSP90 was achieved through geldanamycin and resorcinol derivates. First, western blotting was carried out to confirm the effect of this inhibition. Later, to identify a proteomic profile associated with HSP90 inhibition, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of selected cell lines were performed. Results: The expression of the oncogenic HSP90 client proteins studied was decreased by the inhibitors in the NSCLC cell lines. The oncoproteins drivers EGFR and EML4-ALK showed a strong dependence on HSP90 observed through a high sensitivity of the cell lines HCC827 and H3122 to the inhibition. Therefore, these cell lines were selected with the purpose of identify a proteomic signature linked to HSP90 inhibition by 2D-PAGE. The untreated EGFR positive cell line presented 104 protein spots significantly up-regulated, whereas 80 spots were down-regulated compared to inhibited cell line. Meanwhile, the cell line harboring the EML4-ALK translocation showed 16 spots up-regulated and 5 down-regulated in the untreated versus inhibited cell line. In addition HSP70 overexpression, compared to unprocessed cell lines, was detected after treatments. This feedback, previously reported, confirmed HSP90 inhibition in the two cell lines studied. Conclusions: The evidence of treatment response, in the cell lines studied, was showed through oncogenic client proteins reduction as well as HSP70 induction. The proteomic profiles identified, of HSP90 inhibited and untreated tumor cells, revealed several deregulated pathways involved in the tumorigenesis. Citation Format: Angela Marrugal, Irene Ferrer, Maria Dolores Pastor, Alvaro Quintanal, Antonio Lucena-Cacace, Amancio Carnero, Luis Paz-Ares, Sonia Molina-Pinelo. HSP90 inhibition leads to a differential proteomics profile in non small cell lung cancer cell lines [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 2207. doi:10.1158/1538-7445.AM2017-2207