Anaplastic Lymphoma Kinase Protein Research Articles

PRIMARY PULMONARY ANAPLASTIC LARGE CELL LYMPHOMA IN A YOUNG MALE

SESSION TITLE: Fellows Lung Cancer Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Anaplastic large cell lymphoma (ALCL) is a type of non-Hodgkin lymphoma which is subclassified into primary systemic anaplastic lymphoma kinase (ALK)-positive, primary systemic ALK-negative, primary cutaneous ALCL, and breast implant-associated ALCL (1). ALCL is considered an extremely rare type of primary pulmonary lymphomas (2). We report a case of primary pulmonary ALCL in a young immunocompetent male presenting with dyspnea. CASE PRESENTATION: 43 year-old male with no past medical history presented to the emergency department with dyspnea, fatigue and lower extremity swelling which have been worsening over a month. Physical exam showed 3+ pitting edema bilaterally and bilateral decreased breath sound. Laboratory workup showed an elevate d-dimer and an elevated absolute monocytes. Chest X-ray showed numerous nodules throughout the lungs. CT angiogram of the chest, abdomen and pelvis showed a large infiltrative opacity extending from the right hilum to the pleural surface in the right lower lobe, with some extension into the central right middle lobe and innumerable pulmonary and hepatic metastases and lymphadenopathy in the thoracic, abdominal and pelvic lymph node chains. Tumor markers were negative, with mildly elevated CA-125. A right supraclavicular lymph node biopsy showed necrotizing lymphadenitis with no evidence of metastatic disease. Bronchoscopy revealed an extensive obstruction of the bronchial lumen with an endobronchial lesion in the right lower lobe bronchus. Tissue biopsy was consistent with anaplastic large cell lymphoma. Patient was critically ill in the intensive care unit and was intubated due to acute respiratory failure. He had a poor prognosis due to the extensive metastasis of his ALCL. DISCUSSION: ALCL is characterized by large lymphoid cells with pleomorphic nuclei, and uniform CD30/Ki-1 expression (2). The most common form of ALCL presents as primary cutaneous lymphoma and may secondarily involve the lungs. However, primary pulmonary ALCL is extremely rare with 10 published cases between 1990 and 2015 (3). ALK protein expression is an important prognostic indicator for ALCL as ALK-positive lymphomas have a favorable prognosis with a much higher 5-year survival rate (2). The diagnosis of ALCL includes histopathology and immunohistochemistry staining, with strong immunoreactivity for CD30. Our patient had a primary pulmonary ALCL which was ALK-negative, indicating poor overall prognosis, with a strong immunoreactivity for CD30 and Ki-1. The treatment of primary pulmonary ALCL has not been standardized. However, first-line therapy includes anthracycline-based regimen, which is the standard treatment for non-Hodgkin lymphomas. CONCLUSIONS: Our case underlines the importance of maintaining a high index of awareness for the occurrence and impact of the recognition of primary pulmonary ALCL and its implications on the clinical course of critically ill patients. Reference #1: Laurent, Camille; Haioun, Corinne; Brousset, Pierre; Gaulard, Philippe (1 September 2018). “New insights into breast implant-associated anaplastic large cell lymphoma”. Current Opinion in Oncology. 30 (5): 292–300. Reference #2: J.-F. Cordier, E. Chailleux, D. Lauque et al., “Primary pulmonary lymphomas; a clinical study of 70 cases in nonimmunocompromised patients,” CHEST, vol. 103, no. 1, pp. 201–208, 1993. Reference #3: Q. Zhao, Y. Liu, H. Chen et al., “Successful chemo-radiotherapy for primary anaplastic large cell lymphoma of the lung: A case report and literature review,” American Journal of Case Reports, vol. 17, pp. 70–75, 2016. DISCLOSURES: No relevant relationships by Obed Adarkwah, source=Web Response No relevant relationships by Raheel Anwar, source=Web Response No relevant relationships by Louis Gerolemou, source=Web Response No relevant relationships by Wael Kalaji, source=Web Response No relevant relationships by jad sargi, source=Web Response No relevant relationships by Viswanath Vasudevan, source=Web Response

Impact of Pre-Analytical Conditions on the Antigenicity of Lung Markers: ALK and MET.

Diagnostic assays for molecular alterations highly correlated with prognosis, predictive efficacy or safety of therapeutics are valuable clinical tools and in some cases approved as companion diagnostics (CDx) by the Federal Food and Drug Administration. For example, assays that determine echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation status have been approved as CDx assay for therapies that target this molecular alteration. Characterizing the parameters that may compromise diagnostic accuracy for molecular biomarkers is critical for optimal patient care. To investigate the impact of pre-analytical handling and processing of tumor tissue on commonly used diagnostic immunohistochemistry-based assays for ALK and mesenchymal epithelial transition protein [c-mesenchymal epithelial transition (c-MET)], we investigated the effects of cold ischemia, fixative type, fixation time, and cut-slide age on staining consistency and intensity using human lung xenograft tumor tissue. Cold ischemia times for up to 5 to 6 hours for c-MET or ALK, respectively had minimal impact on staining. The optimal fixation conditions for both assays were found to be at least 6 hours and up to 48 hours for c-MET or 72 hours for ALK, in 10% neutral buffered formalin and Zinc formalin. The ALK antigen demonstrated marked staining intensity differences across non-neutral buffered formalin fixative types and times. Finally, cut-slide age influenced assay performance for both ALK and c-MET, with maximum stability observed when cut slides were stored at ambient temperatures (30°C) for no longer than 3, and 5 months, respectively. This study highlights the potential for pre-analytical factors to confound diagnostic test result interpretation.

Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK positive cancer resistance

EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on 293T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that can potentially overcome resistance in cancer targeted therapy.

Density Functional Theory and Molecular Simulation Studies for Prioritizing Anaplastic Lymphoma Kinase Inhibitors.

Targeting anaplastic lymphoma kinase (ALK) is one of the important treatment strategies for the treatment of non-small cell lung cancer (NSCLC). In the present perspective, multidimensional approaches were used for the identification of ALK inhibitors. Initially, an e-pharmacophore model was generated using the PHASE algorithm and was used as a 3D query to screen 468,200 molecules of ASINEX database. Prior to the screening process, the model was evaluated for its significance and the ability to differentiate actives from inactives, using enrichment analysis. Subsequently, the hierarchical docking protocol and binding free energy calculations were instigated using GLIDE algorithm and Prime module, respectively. Further, the pharmacokinetic/pharmacodynamics (PK/PD) properties and toxicities of the hit compounds were envisaged respectively using QikProp program, Osiris explorer, and Protox-II algorithm. These approaches retrieved two hits namely BAS 00137817 and BAS 00680055 with acceptable absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and higher affinity towards ALK protein. Additionally, density functional theory calculations and molecular dynamics simulations were performed to validate the inhibitory activity of the lead compounds. It is noteworthy to mention that all the hits constitute of particular scaffolds which play a major role in the downregulation of some ALK-positive lung cancer pathways. We speculate that the outcomes of this research are of substantial prominence in the rational designing of novel and efficacious ALK inhibitors.

The prognostic roles of and correlation between ALK and MYCN protein expression in neuroblastoma

AimsTo investigate the relations between anaplastic lymphoma kinase (ALK) and v-myc myelocytomatosis viral related oncogene neuroblastoma derived homolog (MYCN) protein expression and their prognostic roles in neuroblastoma tumours.MethodsSixty-one neuroblastoma tumours...

Abstract 2982: Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers

Abstract Introduction: Since the discovery of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein like-4 (EML4) fusion protein, ALK tyrosine kinase inhibitors have been shown to have great anti-tumor activities against ALK rearranged non-small cell lung cancers (NSCLC). As previously reported, the EML4-ALK fusion proteins induce constitutive phosphorylation of ALK via the trimeric coiled-coil domain within EML4. ALK F1174L mutations known in neuroblastoma, can induce autophosphorylation without dimerization. We hypothesize that monomerization of ALK fusion proteins decreased tumorigenesis via competitive dissociation of the trimeric coiled-coil domain of EML4. Methods: We established Ba/F3 cells stably expressing wild-type (wt) ALK intracellular domain or F1174L mutant (mu) with using iDimerize system (Takara Bio). Using this system, monomer or dimer of ALK fusion proteins were controlled by adding AP20187 (Takara Bio). These Ba/F3 cells were subcutaneously injected into BALB nu/nu mice followed by intraperitoneal administration of AP20187 or Mock intermittently. A chemically synthesized peptide composed of amino acid sequences of coiled-coil domain within EML4, was administered for H3122 (EML4-ALK) or A549 (KRAS) cells and the viabilities were analyzed after 24 hours. In order to demonstrate the specificity of the peptide, a synthesized peptide in which two amino acids were substituted was also used as a control. Each peptide was solubilized in DMSO and the peptide concentration was optimized. Results: We successfully established the Ba/F3 stably expressing fusion-ALKwt, fusion-ALKmu under continuous administration of AP20187. Subsequently, ALK proteins were depolymerized by withdrawal of AP20187. The number of cells expressing ALKwt were decreased while the same of ALKmu were increased. The protein expression of phosphorylation of ALK or downstream survival signaling proteins were weakened after 24hours from AP20187 withdrawal. In fusion-ALKwt cells, tumor xenograft was developed in every mice with AP20187 (n=6) administration, while tumor was not developed in Mock controls (n=6). In fusion-ALKmu cells, tumor xenograft was developed in all mice with or without AP20187 administration (n=12). In vitro assay, cell growth of H3122 was 40% decreased by adding coiled-coil peptides, while increased cell growth was observed in control peptide groups and A549 cells. Conclusion: Monomerization of ALK intracellular domain induced cell death and suppressed tumorigenesis. The coiled-coil peptide specifically suppressed the activity of EML4-ALK cell line. Monomerization of ALK fusion proteins via coiled-coil peptide is a promising therapeutic strategy for ALK rearranged NSCLC. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2982.

Abstract 4648: Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells

Abstract Background: Chromosomal inversions involving Anaplastic lymphoma kinase (ALK) and Echinoderm Microtubule Associated Protein Like 4 (EML4) generate a fusion protein EML4-ALK with the constitutive ALK kinase activity in non-small cell lung carcinoma (NSCLC). The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. Material and Method: We investigate the activities of EML4-ALK in mortal and immortalized normal human cells. Results: The expression of EML4-ALK in normal, mortal human fibroblasts caused, through its ALK kinase activity, the early induction of cellular senescence with senescence-associated β-galactosidase activity, upregulation of p16INK4A and p21WAF1, telomere shortening and fusions, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts, the cells showed accelerated proliferation and anchorage-independent growth, revealing its transformation activity. No chromosome aberration, no mutation or loss of p53, nor impairment of the p16INK4A response was associated with this transformation, likely reflecting clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. EML4-ALK was also able to transform hTERT-immortalized human bronchial epithelial cells, a cell type relevant to NSCLC. Conclusions: Our data not only validate that EML4-ALK functions as an oncogene in human cells, but also suggest that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. This study also provides the isogenic pairs of human cell lines with and without EML4-ALK as an in vitro model for screening and testing candidate drugs. Citation Format: Akihiko Miyanaga, Izumi Horikawa, Masaru Matsumoto, Takahiro Oike, Jessica Beck, Hiromi Tanaka, Ana I. Robles, Masahiro Seike, Akihiko Gemma, Curtis C. Harris. Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4648.

Anticancer Screening of the Phytochemicals Present in the Medicinal Plant Vitex Negundo Against Mutant Anaplastic Lymphoma Kinase (ALK) Protein: An In-Silico Approach

The lymphomas are a heterogeneous group of cancer of the lymphocytes and the lymphatic system and accounts for up to 3% of all malignancies.1 Most of the drugs currently used for the treatment of lymphoma produce various side effects, hence in this study, we focus on natural compounds, obtained from the medicinal plant Vitex negundo, which exhibits selective toxicity against cancer cells. The objective of this research was to formulate the binding energies and interaction of selected phytochemicals present in the medicinal plant Vitex negundo2 against anaplastic lymphoma kinase protein, which is overexpressed in an anaplastic large cell lymphoma.3, 4,5 The structure of mutant human anaplastic lymphoma kinase protein was retrieved from the Protein Data Bank (PDB ID:4ANL ) and the 3D chemical structure of the phytochemicals present in the medicinal plant Vitex negundo was obtained from the PubChem database. Molecular docking study was performed for these natural compounds to evaluate and analyze their anti-lymphoma-cancer activity. A total of 16 compounds present in Vitex negundo, based on a comprehensive literature survey was selected for this molecular screening. Molecular docking analysis was carried out by Molegro Virtual Docker software, to screen the 16 chosen compounds and rank them according to their binding affinity towards the site of interaction of the oncoprotein, anaplastic lymphoma kinase. Out of the 16 screened phytocompounds, only 4 compounds showed promising interactions against the oncoprotein ALK (4ANL). 6’-p-hydroxybenzoyl mussaenosidic acid exhibited a very good binding with a molecular docking score of -127.723 kcal/mol, ranking first among the compounds screened. This was followed by Betulinic acid, Viridiflorol and protocatechuic acid with molecular docking scores of -95.596 kcal/mol, -76.1648 kcal/mol and -63.0854 kcal/mol and - respectively. The docking scores from the above study shows that the phytocompounds present in Vitex negundo extract exhibits an effective inhibitory effect against anaplastic lymphoma kinase protein that is over expressed in lymphoma.

Implementation of functional precision medicine for anaplastic lymphoma kinase-rearranged non-small lung cancer

Anaplastic lymphoma kinase (ALK)-rearranged kinase drives non-small cell lung cancer (NSCLC) in 3–7% of patients and treatment consists of a broad range of approved inhibitors which are administered sequentially in case of resistance developing invariably after approximately 1 year of therapy. Besides the former standard tyrosine kinase inhibitor (TKI) crizotinib, the second-line ALK inhibitors alectinib, ceritinib, brigatinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find individual schemes of ALK inhibitors therapy which provide the best benefit for the patients. A host of ALK fusion partners, rearrangement variants and ALK mutations prevent a direct correlation of ALK alterations and sensitivity to specific TKIs within the framework of genomic precision medicine. However, recurrent ALK-positive NSCLC is an aggressive disease resulting in accumulation of tumor cells in pleural effusions which may be collected for in vitro sensitivity testing in a manner designated as functional precision medicine. Provided that these cells are present in sufficient numbers, they can be exposed to all possible drug candidates and their chemosensitivity tested in short-term proliferation assays. In addition to ALK-directed TKIs, cytotoxic drugs and inhibitors of non-target bypassing pathways may be included in the tests. In contrast to other trials in genome-guided precision medicine, a range of suitable therapeutics is available for modified ALK proteins. In summary, ALK-positive NSCLC with pleural effusions offer a unique model to develop, test and validate concepts of functional precision medicine.

A phase I clinical study to evaluate the safety, tolerability, pharmacokinetics (PK), and antitumor activity of FN-1501 monotherapy in patients with advanced solid tumors.

TPS3150 Background: Receptor tyrosine kinases (RTK), a group of transmembrane proteins, are responsible for growth factor signaling transduction in normal cellular functions. Abnormal RTK functions are associated with human tumorigenesis. FMS-like tyrosine kinase 3 (FLT3) belongs to the type III receptor tyrosine kinase family and plays a well-established role in normal growth and differentiation of hematopoietic precursor cells. FLT3 mutations have been reported to occur in approximately 30% newly diagnosed AML patients. The internal tandem duplications mutation (FLT3/ITD) is the major mutation and correlated with more aggressive progress and poor prognosis. FN-1501 is an inhibitor of various tyrosine kinases such as cyclin-dependent kinase 4/6(CDK4/6), platelet-derived growth factor receptor (PDGFR), KIT protein, anaplastic lymphoma kinase (ALK) and RET protein, particularly potent on FLT3. The preclinical data generated from biochemical, cell based and animal in vivo studies suggest that FN-1501 as a single agent could offer cancer patients clinical benefit by inhibiting multiple tyrosine kinases including FLT3, PDGFR, KIT, ALK, and RET. Methods: This is a Phase1, open label, multicenter, dose-escalation study that will evaluate the safety, pharmacokinetics (PK), and preliminary efficacy of FN-1501 in up to 33 cancer patients with solid tumors. There is a dose escalation phase that will be followed by an expansion cohort. The dose escalation phase utilizes a standard “3 + 3” design where doses of FN-1501 will be escalated up to the Maximum-Tolerated Dose (MTD) or until the Recommended Phase 2 dose (RP2D) is identified. Once the MTD or RP2D dose is identified, an expansion cohort including patients with hematologic malignancies will be enrolled to further evaluate the safety and efficacy of FN-1501. Key exploratory analyses will include an evaluation of safety and efficacy and levels of expression and/or amplification of FLT3 mutations. As of February 8, 2019, cohorts 1 and 2 have been completed without a dose limiting toxicity (DLT). A total of 11 patients have been treated. Enrollment to cohort 3 is on-going. Clinical trial information: NCT03690154.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion

BackgroundGenetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer.MethodsIn 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription–PCR, respectively.ResultsCompared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion–positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion–negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion.ConclusionsThis study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

A Comparative Study of Cell Block versus Biopsy for Detection of Epidermal Growth Factor Receptor Mutations and Anaplastic Lymphoma Kinase Rearrangement in Adenocarcinoma Lung.

Background:Lung cancer is a leading cause of deaths attributed to cancer worldwide. Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement are commonly found in patients of adenocarcinoma lung against, which targeted therapy is available. In this era of personalized medicine, it is a rationale to detect these molecular alterations in cases of lung carcinomas.Aims:The objectives were to compare the diagnostic efficacy of cytological samples for the detection of EGFR and ALK protein expression using immunocytochemistry in nonsmall cell lung carcinoma.Materials and Methods:We compared 22 cell blocks and biopsies for the detection of EGFR and ALK protein expression by immunohistochemistry (IHC). EGFR IHC was performed using EGFR Receptor (E746-A750 del Specific) (6B6) monoclonal antibody and ALK IHC was done using Ventana anti-ALK (D5F3) monoclonal primary antibody.Results:Two cases were found to be positive; 20 cases were negative for EGFR IHC both in biopsies and cell blocks. ALK IHC was positive in one case; negative in 21 cases. The results of IHC were also concordant for biopsies and cell blocks. The sensitivity and specificity were 100% for immunocytochemical detection of ALK and EGFR in cell blocks with respect to biopsies.Conclusion:We conclude that cell blocks can serve as a potential substitute for biopsies for detection of EGFR and ALK protein by immunocytochemistry, whenever patient presents with effusion and biopsy cannot be done or when tissue is not adequate.

Evaluation of anaplastic lymphoma kinase protein immunohistochemical expression in bronchogenic carcinoma

Evaluation of anaplastic lymphoma kinase protein immunohistochemical expression in bronchogenic carcinoma

Expanding the Molecular Characterization of Thoracic Inflammatory Myofibroblastic Tumors beyond ALK Gene Rearrangements

IntroductionHalf of inflammatory myofibroblastic tumors (IMTs) regardless of anatomic location harbor anaplastic lymphoma kinase gene (ALK) rearrangements and overexpress anaplastic lymphoma kinase protein. The wide application of next-generation sequencing and the clinical benefit to tyrosine kinase inhibitors have opened new opportunities for investigation of ALK-negative IMTs. MethodsIn this study, we have investigated a series of pediatric and adult thoracic IMTs for abnormalities in a wide spectrum of actionable kinases by applying a variety of molecular and next-generation sequencing techniques, including fluorescence in situ hybridization (FISH), targeted RNA sequencing, and NanoString assay. ResultsThere were 33 patients with thoracic IMTs, including five children; their mean age was 37. The tumors showed a monomorphic spindle cell phenotype, except for one with an epithelioid morphologic pattern and moderate to severe atypia. By immunohistochemistry, 24 tumors were ALK positive, and 19 of the 24 showed ALK rearrangements and one ret proto-oncogene gene (RET) rearrangement by FISH. RNA sequencing was performed in the remaining four cases lacking ALK abnormalities by FISH, revealing ALK fusions involving tropomyosin 4 gene (TMP4) and echinoderm microtubule associated protein like 4 gene (EML4) as partner in three cases. NanoString assay was performed in the remaining case, revealing ALK alternative transcription initiation (ALKATI). Nine cases lacking ALK abnormalities were further tested by FISH or targeted RNA sequencing, revealing ROS1 rearrangement in six cases and ETS variant 6 gene (ETV6)–neurotrophic receptor tyrosine kinase 3 gene (NTRK3) fusion in three cases, respectively. ConclusionsBy using a battery of complementary molecular techniques, we have shown that all the thoracic IMTs harbored a tyrosine kinase abnormality, with 30% involving a kinase gene other than ALK, including ROS1, NTRK3, and RET gene fusions. We have also described for the first time ALKATI-induced ALK oncogenic activation in IMTs.

Discordance between FISH, IHC, and NGS Analysis of ALK Status in Advanced Non–Small Cell Lung Cancer (NSCLC): a Brief Report of 7 Cases

BACKGROUND: Anaplastic lymphoma kinase (ALK) rearrangement represents a landmark in the targeted therapy of non–small cell lung cancer (NSCLC). Immunohistochemistry (IHC) is a sensitive and specific method to detect ALK protein expression, possibly an alternative to fluorescence in situ hybridization (FISH). In this study, the concordance of FISH and IHC to determine ALK status was evaluated, particularly focusing on discordant cases. MATERIALS AND METHODS: ALK status was tested by FISH and the IHC validated method (Ventana ALK (D5F3) CDx Assay) in 95 NSCLCs. Discordant cases were analyzed also by next-generation sequencing (NGS). The response to crizotinib of treated patients was recorded. RESULTS: Seven (7.3%) discordant cases were ALK FISH positive and IHC negative. They showed coexistent split signals pattern, with mean percentage of 15.4%, and 5′ deletions pattern, with mean percentage 31.7%. Two cases had also gene amplification pattern. In three cases (42.8 %), the polysomy was observed. The NGS assay confirmed IHC results. In these patients, the treatment with crizotinib was ineffective. CONCLUSIONS: In our discordant cases, a coexistent complex pattern (deleted, split, and amplified/polysomic) of ALK gene was observed by FISH analysis. These complex rearranged cases were not detectable by IHC, and it could be speculated that more complex biological mechanisms could modulate protein expression. These data highlight the role of IHC and underscore the complexity of the genetic pattern of ALK. It could be crucial to consider these findings in order to best select patients for anti-ALK treatment in daily clinical practice.

Identification of Novel Pathways and Small Molecules Able to Down Regulate Oncogenes Expression By Compounds Screening Approaches in Acute Leukemia and Neuroblastoma Cells

Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, resistance to ALK inhibitors is a ubiquitous problem in ALK-expressing cell lines as well as treated patients. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. Previous reports have suggested that mechanism of resistance is mediated by mutations in the ALK kinase domain impairing binding of an inhibitor to an ALK protein. Thus, new treatment modalities are urgently needed to sensitize patients to crizotinib thereby improving the management of hematological or solid malignancies harboring ALK mutations. To identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, we implemented a high throughput chemical screen in 4 NB-derived cell lines, using a curated library of ~450 compounds. In the compounds screening, JAK-STAT kinase inhibitor (cucurbitacin I) was the most discriminatory with regard to sensitivity for ALK-mutated cell lines. Since Gamma cytokine JAK/STAT system as a target in the treatment of T-cell or myeloid malignancies, we analyzed the cytotoxicity of cucurbitacin I antitumor effect using an MTT assay revealed cucurbitacin I to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by cucurbitacin I (IC50values:T-ALL lines, ICH-TALL-UK, 103.2 nM; MOLT-14, 35.6 nM; KCMC-T, 134.2 nM; Jurkat, 77 nM; ICH-TALL-SM, 47.7 nM; MOLT-4, 22.2 nM. AML lines, HEL, 66.7 nM; Kasumi-3, 4.5 nM; KG-1, 53.6 nM; THP-1, 337.2 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of cucurbitacin I. In NB cell lines harboring F1174L or R1275Q-mutated ALK, crizotinib combined with cucurbitacin I enhanced tumor responses and showed synergistic cytotoxicity.Although crizotinib and cucurbitacin I alone or combination therapy (cucurbitacin I + crizotinib) did not result in decreased viability over control compared with vehicle, the combination therapy in all of 6 cell lines with ALK aberrations and 10 of 13 ALK wild-type cell lines with MYCN amplification or 11q LOH was more effective than vehicle, crizotinib alone, and cucurbitacin I alone. Analysis of downstream signalling through MAPK, AKT and STAT3 pathways showed that NIH3T3 cells stably expressed F1174L mutated ALK or TGW cells harboring R1275Q-mutated ALK, expressed lower levels of pERK, pAKT and pSTAT3 in combination therapy compared with cells treated with cucurbitacin I or crizotinib alone. These findings may provide a indication that the combination of low-dose ALK and STAT3 inhibitors may be benefitical for the treatment of NB, by enhancing efficacy while reducing toxicity. In conclusion, our studies suggested that NB, T-ALL or AML cell lines also exhibited potent cytotoxic responses to the cucurbitacin I and the combination of ALK and JAK-STAT inhibitors could be a valuable therapeutic option for ALK mutated malignancies such as high-risk NB with potential clinical application. DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 Generation of Npm1-Alk in Transplantable Murine Hematopoietic Stem Cells Accurately Models ALK-Positive Lymphoma in Recipients

Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies.Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3‘ ALK kinase domain to the 5‘ oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients.Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients.We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. DisclosuresNo relevant conflicts of interest to declare.

Translocation-Related Sarcomas.

Chromosomal translocations are observed in approximately 20% of soft tissue sarcomas (STS). With the advances in pathological examination technology, the identification of translocations has enabled precise diagnoses and classifications of STS, and it has been suggested that the presence of and differences in translocations could be prognostic factors in some translocation-related sarcomas. Most of the translocations in STS were not regarded as targets of molecular therapies until recently. However, trabectedin, an alkylating agent, has shown clinical benefits against translocation-related sarcoma based on a modulation of the transcription of the tumor’s oncogenic fusion proteins. Many molecular-targeted drugs that are specific to translocations (e.g., anaplastic lymphoma kinase and tropomyosin kinase related fusion proteins) have emerged. The progress in gene technologies has allowed researchers to identify and even induce new translocations and fusion proteins, which might become targets of molecular-targeted therapies. In this review, we discuss the clinical significance of translocation-related sarcomas, including their diagnoses and targeted therapies.

RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance

BackgroundRearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma.MethodsWe selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series.ResultsCases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript.ConclusionsThe comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.

P1.09-22 Detection of ALK Gene Rearrangement in FFPE Tissues of Non-Small Cell Lung Cancer Using in Situ RNA Hybridization

Approximately 4-7% of non-small cell lung cancer (NSCLC) harbor anaplastic lymphoma kinase (ALK) gene rearrangements which lead to overexpression of ALK fusion protein and constitutive activation of the kinase. The tumor with ALK gene rearrangements is sensitive to its inhibitor, so identification of this molecular feature from the patients accurately is important. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are conventional methods for testing ALK gene rearrangement and protein overexpression respectively; however it is challenge for distinguishing some rare variations by FISH, or determining the equivocal stain by IHC. A morphology related method under bright field about rearranged mRNA expression (RNAscope) of ALK gene fusion was developed, which is helpful to realize the biological behavior of ALK gene, together with FISH (break apart FISH probe kit, Abbott) and IHC (D5F3 clone, Ventana). We explored 7 cases of formalin-fixed paraffin-embedded (FFPE) samples from NSCLC patients by RNAscope® 2.5 Red assay Three of seven samples were found with ALK positive by RNAscope, which matched with FISH and IHC results. In these 3 ALK positive samples, exon 19-29 of ALK gene transcribed RNA signals were revealed by probe-Hs-ALK E1-E18 (score 1 to 2 in >50% cells), whereas exon 1-18 transcribed RNA signals were not shown by probe-Hs-ALK E19-E29, which confirmed by FISH results (with break apart signals). In 2 of 4 ALK RNAscope negative cases, ALK RNA signals were detected by both probe-Hs-ALK E1-E18 (score 2) and probe-Hs-ALK E19-E29 (score 2) in small regions (showed a small number of signals), which may indicate that ALK gene amplification and also confirmed by FISH results (with more than 2 fusion signals). And these 2 typical ALK RNAscope negative samples were considered close to be disomy by FISH. Our results demonstrate that RNAscope® 2.5 Red assay is a reliable and precise method to detect ALK mRNA expression caused by ALK gene fusion and also a one to discover the ALK gene amplification as well in FFPE samples of NSCLC, even though latter’s clinical significant is not clear. RNAscope® 2.5 Red assay is a both sensitive and specific method to provide information on the spatial distribution of ALK gene status and morphologic characteristic of tumors simultaneously under the bright field microscope.