crizotinib and Pleural-Effusion--Malignant

Approximately 5-7% of non-small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics.

Topics: Adult; Aged; Aged, 80 and over; Anaplastic Lymphoma Kinase; Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Proliferation; Cohort Studies; Crizotinib; DNA, Neoplasm; Early Detection of Cancer; ErbB Receptors; Female; Formaldehyde; Gene Rearrangement; HEK293 Cells; Humans; Lung Neoplasms; Male; Microtubule-Associated Proteins; Middle Aged; Paraffin Embedding; Pleural Effusion, Malignant; Serine Endopeptidases; Tissue Fixation

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Anaplastic Lymphoma Kinase; Antineoplastic Agents; Carbazoles; Cell Separation; Crizotinib; DNA; Drug Resistance, Neoplasm; Exons; Female; Gene Amplification; Gene Deletion; Gene Expression Profiling; Genes, erbB-1; High-Throughput Nucleotide Sequencing; Humans; Immunomagnetic Separation; Keratins; Leukocyte Common Antigens; Liquid Biopsy; Lung Neoplasms; Male; Middle Aged; Mutation; Neoplastic Cells, Circulating; Oncogene Proteins, Fusion; Piperidines; Pleural Effusion, Malignant; Protein Kinase Inhibitors

Cell-free DNA (cfDNA) has been a research hotspot in molecular tumor profiling. In advanced gastric cancer patients, malignant pleural effusion (MPE) and ascites provide a wealth of tumor cells that can be investigated. Here we conducted next-generation sequencing (NGS) on matched cfDNA from plasma, MPE and ascites from a stage-IV gastric cancer patient to identify potential therapeutic targets. In all three samples, we detected an amplification in the cellular-mesenchymal to epithelial transition factor (MET) gene, a truncation mutation in SMAD3 (p.R368X), and four ataxia telangiectasia-mutated gene (ATM) variants, including a missense mutation (p.E2351A), an in-frame deletion (p.NPAVIM2353delinsK), a frame-shift deletion (p.D1758fs) and an ATM- BPI fold containing family B member 1 (BPIFB1) gene fusion. In contrast, we detected amplification of TEK only in malignant ascites. The patient was subjected to Crizotinib to counter MET amplification. Our study demonstrates high accordance in mutational spectra of matched cfDNA from plasma, MPE and ascites, and suggests that it is feasible to utilize these tumor sources in clinical decision-making.

Topics: Ascites; Biomarkers, Tumor; Circulating Tumor DNA; Crizotinib; DNA Mutational Analysis; Gene Amplification; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Pleural Effusion, Malignant; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Stomach Neoplasms

The mechanism of the first-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) acquired resistance included T790M mutation, cellular-mesenchymal to epithelial transition factor (MET) or EGFR amplification, PIK3CA mutation, and transformation to small cell lung cancer. MET amplification accounted for only about 5% of the resistance cases.. Few report detected MET amplification in pleural effusion. Here, we reported 2 lung adenocarcinoma cases with MET amplification in pleural effusion rapidly responded to crizotinib after EGFR-TKIs acquired resistance.. Biopsy via bronchoscopy, next-generation sequencing (NGS) in pleural effusion.. EGFR-TKIs (Icotinib), MET inhibitor crizotinib.. After a progression-free survival of 9 months and 23months, respectively, both cases progressed accompanying with pleural effusion. Results of NGS in pleural effusion showed MET amplification (2-3 times) in both cases. The 2 patients were treated with a MET inhibitor crizotinib and rapidly responded.. MET amplification in pleural effusion could predict a perfect response to crizotinib after EGFR-TKIs acquired resistance, even only a low times gene amplification.

Topics: Adenocarcinoma; Aged; Crizotinib; Drug Resistance, Neoplasm; Female; Gene Amplification; Humans; Lung Neoplasms; Male; Middle Aged; Pleural Effusion, Malignant; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines

The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs.. MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists.. We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs.. We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

Topics: Aged; Antineoplastic Agents; Cisplatin; Crizotinib; Dose-Response Relationship, Drug; Drug Combinations; Drug Screening Assays, Antitumor; Humans; Immunomagnetic Separation; Male; Middle Aged; Pemetrexed; Pleural Effusion, Malignant; Precision Medicine; Primary Cell Culture; Pyrazoles; Pyridines; Tumor Cells, Cultured

The echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients with non-small cell lung cancer. To the best of our knowledge, there are only three cell lines harboring the EML4-ALK fusion gene, which have contributed to the development of therapeutic strategies. Therefore, we tried to establish a new lung cancer cell line harboring EML4-ALK.. A 61-year-old Japanese female presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was treated with chemotherapy, her disease progressed with massive pleural effusion. Because the EML4-ALK rearrangement was found in a biopsied specimen using fluorescence in situ hybridization, she was treated with crizotinib. She did well for 3 months.. Tumor cells were obtained from the malignant pleural effusion before treatment with crizotinib. Cells continued to proliferate substantially for several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein and genes were identified in ABC-11 cells using fluorescence in situ hybridization and immunohistochemistry, respectively. ABC-11 cells were sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and AP26113), as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Phosphorylated ALK protein and its downstream signaling were suppressed by treatment with crizotinib in western blotting. Furthermore, we could transplant ABC-11 cells subcutaneously into BALB/c nu/nu mice.. We successfully established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene. This cell line could contribute to future research of EML4-ALK-positive lung cancer both in vivo and in vitro.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Cell Cycle Proteins; Cell Line, Tumor; Crizotinib; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Middle Aged; Oncogene Proteins, Fusion; Pleural Effusion, Malignant; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Receptor Protein-Tyrosine Kinases; Serine Endopeptidases; Signal Transduction; Sulfones