RESUMO
Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells.
Assuntos
Cinesinas , Quinases Relacionadas a NIMA , Proteínas de Fusão Oncogênica , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genética , Humanos , Fosforilação , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Mesoderma/metabolismo , Mesoderma/patologia , Linhagem Celular Tumoral , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Most EML4-ALK rearrangement non-small cell lung cancer (NSCLC) patients inevitably develop acquired drug resistance after treatment. The main mechanism of drug resistance is the acquired secondary mutation of ALK kinase domain. L1196M and G1202R are classical mutation sites. We urgently need to understand the underlying molecular mechanism of drug resistance to study the therapeutic targets of mutant drug-resistant NSCLC cells. The silent information regulator sirtuin1 (SIRT1) can regulate the normal energy metabolism of cells, but its role in cancer is still unclear. In our report, it was found that the SIRT1 in EML4-ALK G1202R and EML4-ALK L1196M mutant drug-resistant cells was downregulated compared with EML4-ALK NSCLC cells. The high expression of SIRT1 was related to the longer survival time of patients with lung cancer. Activation of SIRT1 induced autophagy and suppressed the invasion and migration of mutant cells. Further experiments indicated that the activation of SIRT1 inhibited the phosphorylation level of mTOR and S6K by upregulating the expression of AMPK, thus activating autophagy. SIRT1 can significantly enhanced the sensitivity of mutant cells to crizotinib, improved its ability to promote apoptosis of mutant cells, and inhibited cell proliferation. In conclusion, SIRT1 is a key regulator of drug resistant in EML4-ALK L1196M and G1202R mutant cells. SIRT1 may be a novel therapeutic target for EML4-ALK drug resistant NSCLC.
RESUMO
Although ALK tyrosine kinase inhibitors (ALK-TKIs) have shown remarkable benefits in EML4-ALK positive NSCLC patients compared to conventional chemotherapy, the optimal sequence of ALK-TKIs treatment remains unclear due to the emergence of primary and acquired resistance and the lack of potential prognostic biomarkers. In this study, we systematically explored the validity of sequential ALK inhibitors (alectinib, lorlatinib, crizotinib, ceritinib and brigatinib) for a heavy-treated patient with EML4-ALK fusion via developing an in vitro and in vivo drug testing system based on patient-derived models. Based on the patient-derived models and clinical responses of the patient, we found that crizotinib might inhibit proliferation of EML4-ALK positive tumors resistant to alectinib and lorlatinib. In addition, NSCLC patients harboring the G1269A mutation, which was identified in alectinib, lorlatinib and crizotinib-resistant NSCLC, showed responsiveness to brigatinib and ceritinib. Transcriptomic analysis revealed that brigatinib suppressed the activation of multiple inflammatory signaling pathways, potentially contributing to its anti-tumor activity. Moreover, we constructed a prognostic model based on the expression of IL6, CXCL1, and CXCL5, providing novel perspectives for predicting prognosis in EML4-ALK positive NSCLC patients. In summary, our results delineate clinical responses of sequential ALK-TKIs treatments and provide insights into the mechanisms underlying the superior effects of brigatinib in patients harboring ALKG1269A mutation and resistant towards alectinib, lorlatinib and crizotinib. The molecular signatures model based on the combination of IL6, CXCL1 and CXCL5 has the potential to predict prognosis of EML4-ALK positive NSCLC patients.
Assuntos
Adenocarcinoma de Pulmão , Antineoplásicos , Neoplasias Pulmonares , Proteínas de Fusão Oncogênica , Compostos Organofosforados , Inibidores de Proteínas Quinases , Pirimidinas , Humanos , Compostos Organofosforados/uso terapêutico , Compostos Organofosforados/farmacologia , Pirimidinas/uso terapêutico , Pirimidinas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Animais , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Prognóstico , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Lactamas/uso terapêutico , Carbazóis/uso terapêutico , Carbazóis/farmacologia , Sulfonas/uso terapêutico , Sulfonas/farmacologia , Crizotinibe/uso terapêutico , Crizotinibe/farmacologia , Linhagem Celular Tumoral , Piperidinas/uso terapêutico , Piperidinas/farmacologia , Feminino , Camundongos , Inflamação/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Pirazóis/uso terapêutico , Pirazóis/farmacologia , Masculino , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/metabolismo , Proliferação de Células/efeitos dos fármacos , Mutação , Aminopiridinas/uso terapêutico , Aminopiridinas/farmacologiaRESUMO
BACKGROUND: Accumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear. METHOD: High-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients' tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS. RESULTS: Sequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression. CONCLUSION: EML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/uso terapêutico , Tromboplastina/genética , Fator de Transcrição AP-1/uso terapêutico , Proliferação de Células , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/metabolismoRESUMO
BACKGROUND: EML4-ALK gene fusions are oncogenic drivers in non-small cell lung cancer (NSCLC), and liquid biopsies containing EML4-ALK fragments can be used to study tumor dynamics using next-generation sequencing (NGS). However, the sensitivity of EML4-ALK detection varies between pipelines and analysis tools. RESULTS: We developed an R/Bioconductor package, DNAfusion, which can be applied to BAM files generated by commercially available NGS pipelines, such as AVENIO. Forty-eight blood samples from a training cohort consisting of 41 stage IV EML4-ALK-positive NSCLC patients and seven healthy controls were used to develop DNAfusion. DNAfusion detected EML4-ALK in significantly more samples (sensitivity = 61.0%) compared to AVENIO (sensitivity = 36.6%). The newly identified EML4-ALK-positive patients were verified using droplet digital PCR. DNAfusion was subsequently validated in a blinded validation cohort comprising 24 EML4-ALK-positive and 24 EML4-ALK-negative stage IV NSCLC patients. DNAfusion detected significantly more EML4-ALK individuals in the validation cohort (sensitivity = 62.5%) compared to AVENIO (sensitivity = 29.2%). DNAfusion demonstrated a specificity of 100% in both the training and validation cohorts. CONCLUSION: Here we present DNAfusion, which increases the sensitivity of EML4-ALK detection in liquid biopsies and can be implemented downstream of commercially available NGS pipelines. The simplistic method of operating the R package makes it easy to implement in the clinical setting, enabling wider expansion of NGS-based diagnostics.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Receptores Proteína Tirosina Quinases , Biópsia LíquidaRESUMO
The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion gene in non-small-cell lung cancer (NSCLC) was first identified in 2007. As the EML4-ALK fusion protein promotes carcinogenesis in lung cells, much attention has been paid to it, leading to the development of therapies for patients with NSCLC. These therapies include ALK tyrosine kinase inhibitors and heat shock protein 90 inhibitors. However, detailed information on the entire structure and function of the EML4-ALK protein remains deficient, and there are many obstacles to overcome in the development of novel anticancer agents. In this review, we describe the respective partial structures of EML4 and ALK that are known to date. In addition to their structures, noteworthy structural features and launched inhibitors of the EML4-ALK protein are summarized. Furthermore, based on the structural features and inhibitor-binding modes, we discuss strategies for the development of novel inhibitors targeting the EML4-ALK protein.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Pulmão/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
EML4-ALK is an oncogenic fusion present in â¼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/genética , Microtúbulos , Quinases Relacionadas a NIMA/genética , Proteínas de Fusão Oncogênica/genética , Receptores Proteína Tirosina QuinasesRESUMO
BACKGROUND: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. METHODS: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. RESULTS: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. CONCLUSIONS: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Vesículas Extracelulares , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/genética , RNA , Receptores Proteína Tirosina Quinases/genéticaRESUMO
In this article, we describe a spindle cell neoplasm harboring an EML4-ALK gene fusion presenting as an intraosseous vertebral mass with extension into the adjacent soft tissue in a 65-year-old man. Histologically, the lesion was characterized by the presence of monotonous, cytologically bland spindle cells with loose myxoedematous stroma and interspersed areas of amianthoid-like collagen fiber deposition. Immunohistochemistry demonstrated strong diffuse staining for CD34 and S100, with absent immunoreactivity for SOX10. At 1 year of follow-up after resection, there is no evidence of local recurrence or metastatic disease. This case adds to the clinical and pathologic spectrum of the recently described group of kinase fusion-positive spindle cell neoplasms and represents the first reported intra-osseous example. The presence of ALK rearrangement in this lesion represents a potential therapeutic target, if clinically indicated.
Assuntos
Neoplasias de Tecido Conjuntivo/diagnóstico , Proteínas de Fusão Oncogênica/genética , Coluna Vertebral/patologia , Idoso , Antígenos CD34/metabolismo , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Diagnóstico Diferencial , Humanos , Masculino , Neoplasias de Tecido Conjuntivo/diagnóstico por imagem , Neoplasias de Tecido Conjuntivo/genética , Neoplasias de Tecido Conjuntivo/patologia , Fatores de Transcrição SOXE/metabolismo , Coluna Vertebral/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Next-generation sequencing has mostly been used for genotyping cell-free DNA (cfDNA) in plasma. However, this assay has several clinical limitations. We evaluated the clinical utility of a novel polymerase chain reaction-free nanowire (NW)-based plasma cfDNA assay for detecting ALK fusion and mutations. PATIENTS, MATERIALS, AND METHODS: We consecutively enrolled 99 patients with advanced non-small cell lung cancer undergoing a fluorescence in situ hybridization (FISH) test for ALK fusion; ALK-positive (n = 36). The NW-based assay was performed using 50-100 µL of plasma collected at pretreatment and every 8 weeks during ALK inhibitor treatment. RESULTS: There was high concordance between the NW-based assay and the FISH test for identification of ALK fusion (94.9% with a kappa coefficient value of 0.892, 95% confidence interval [CI], 0.799-0.984). There was no difference in the response rate to the first anaplastic lymphoma kinase inhibitor between the ALK-positive patients identified by the NW-based assay and by the FISH test (73.5% vs. 72.2%, p = .931). In the ALK variant analysis, variants 1 and 3 subgroups were detected in 27 (75.0%) and 8 (22.2%) patients, respectively. Among 24 patients treated with crizotinib, variant 3 subgroup was associated with worse median overall survival than variant 1 subgroup (36.5 months; 95% CI, 0.09-87.6 vs. 19.8 months; 95% CI, 9.9-not reached, p = .004]. A serial assessment identified that ALK L1196M resistance mutation emerged before radiologic progression during crizotinib treatment. CONCLUSION: The newly developed simple NW-based cfDNA assay may be clinically applicable for rapid diagnosis of ALK fusion with its variant forms and early detection of resistance. IMPLICATIONS FOR PRACTICE: The authors developed a novel one-step polymerase chain reaction-free nanowire (NW)-based plasma cell-free DNA (cfDNA) assay. This study evaluated the clinical utility of this novel method for the diagnosis of EML4-ALK fusion in advanced non-small cell lung cancer (NSCLC). The NW-based assay and FISH test showed high concordance rate in 99 patients with advanced NSCLC. Serial cfDNA assessment demonstrated this method provided early detection of resistance before radiologic progression during crizotinib treatment. Taken together, plasma cfDNA genotyping by the NW-based cfDNA assay may be useful for the rapid diagnosis of ALK fusion, classifying variants, and early detection of resistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Ácidos Nucleicos Livres , Neoplasias Pulmonares , Nanofios , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ácidos Nucleicos Livres/genética , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated ß-galactosidase (SA-ß-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.
Assuntos
Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/metabolismo , Telomerase/metabolismo , Animais , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Proliferação de Células/genética , Senescência Celular/genética , Dano ao DNA , Modelos Animais de Doenças , Células Epiteliais , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas de Fusão Oncogênica/genética , RNA-Seq , Telomerase/genética , Homeostase do Telômero/genética , TransfecçãoRESUMO
Recently, a novel group of CD34+ and S100+ spindle cell tumors with distinctive stromal and perivascular hyalinization showing recurrent gene fusions involving RAF1, BRAF, NTRK1/2/3, and RET has been identified. ALK rearrangements have been rarely reported in this group of tumors. We report a 24-year-old woman with a 1.5-cm pink mass of the scalp. The tumor was made of spindle cells organized in fascicles or haphazardly arranged in a patternless architecture, with areas of stromal and perivascular hyalinization. The tumor cells diffusely expressed CD34 and S100, without SOX-10 expression. The tumor showed diffuse immunopositivity for ALK. RNA sequencing using next-generation sequencing (NGS) detected an EML4-ALK fusion. This case extends the spectrum of this newly described group of CD34+/S100+ spindle cell tumors at the molecular-genetic level. Dermatopathologists should be aware of this recent entity, as it may fall in the differential diagnosis of many other spindle cell tumors with CD34 expression. NGS-based techniques should be performed when facing spindle cell tumors with similar morphology and immunophenotype. Identification of kinase fusions is essential for the precise classification and better knowledge of these tumors, and for targeted therapy in rare aggressive cases.
Assuntos
Antígenos CD34/metabolismo , Proteínas S100/metabolismo , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/patologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Diagnóstico Diferencial , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hialina/metabolismo , Imuno-Histoquímica/métodos , Proteínas de Fusão Oncogênica , Análise de Sequência de RNA/métodos , Neoplasias de Tecidos Moles/diagnóstico , Células Estromais/metabolismo , Células Estromais/patologia , Recusa do Paciente ao Tratamento , Adulto JovemRESUMO
Gastric mesenchymal tumors are relatively rare, and their molecular pathogeneses are poorly understood, except for gastrointestinal stromal tumor, desmoid, and inflammatory myofibroblastic tumors. We report a case of a gastric mesenchymal tumor with prominent smooth muscle cell differentiation and an echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion. On gross section, the tumor was 26 mm at the largest diameter, well-circumscribed, and located in the submucosal and muscular layers of the stomach wall. Histologically, the tumor comprised intersecting fascicles of spindle cells, non-atypical nuclei, and highly eosinophilic cytoplasm. Myxoid changes were observed focally, but inflammatory infiltrates were only evident in limited areas. Immunochemical staining revealed that the tumor was positive for α-smooth muscle actin and desmin. Diffuse positive staining for h-caldesmon was observed throughout the tumor, which suggested smooth muscle cell differentiation. Intracytoplasmic staining for ALK protein was also observed, and fluorescence in situ hybridization using ALK break-apart probes showed split chromosomal signals. RNA-sequencing analysis identified EML4-ALK fusion transcripts. We concluded that the tumor was a gastric mesenchymal tumor with smooth muscle differentiation based on its distinct differential smooth muscle properties, such as highly eosinophilic cytoplasm and diffuse expression of h-caldesmon. Furthermore, activated ALK may underly the tumor's pathogenesis.
Assuntos
Biomarcadores Tumorais/metabolismo , Leiomioma/diagnóstico , Músculo Liso/patologia , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Gástricas/diagnóstico , Idoso , Diferenciação Celular , Feminino , Humanos , Leiomioma/metabolismo , Leiomioma/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: The echinoderm microtubule-associated protein-like-4 anaplastic lymphoma kinase (EML4-ALK) fusion gene was identified in a subset of non-small cell lung cancer (NSCLC) patients. They responded positively to ALK inhibitors. This study aimed to characterize the mechanisms triggered by EML4-ALK to induce NSCLC transformation. METHODS: HEK293 and NIH3T3 cells were transfected with EML4-ALK variant 3 or pcDNA3.1-NC. H2228 cells were transfected with siRNA-EML4-ALK or siRNA-NC. Cell viability and proliferation were measured by the CCK-8 and EdU methods, respectively. Flow cytometry revealed apoptosis. Gene expression profiles were generated from a signaling pathway screen in EML4-ALK-regulated lung cancer cells and verified by qPCR and Western blotting. The co-immunoprecipitation and immunohistochemistry/ immunofluorescence determined the interaction and colocalization of JAK2-STAT pathway components with EML4-ALK. RESULTS: Microarray identified several genes involved in the JAK2-STAT pathway. JAK2 and STAT6 were constitutively phosphorylated in H2228 cells. EML4-ALK silencing downregulated phosphorylation of STAT6. Expression of EML4-ALK in HEK293 and NIH3T3 cells activated JAK2, STAT1, STAT3, STAT5, and STAT6. In EML4-ALK-transfected HEK293 cells and EML4-ALK-positive H2228 cells, activated STAT6 and JAK2 colocalized with ALK. STAT3 and STAT6 were phosphorylated and translocated to the nucleus of H2228 cells following IL4 or IL6 treatment. Apoptosis increased, while cell proliferation and DNA replication decreased in H2228 cells following EML4-ALK knockdown. In contrast, HEK293 cell viability increased following EML4-ALK overexpression, while H2228 cell viability significantly decreased after treatment with ALK or JAK-STAT pathway inhibitors. CONCLUSIONS: Our data suggest that the aberrant expression of EML4-ALK leads to JAK2-STAT signaling pathway activation, which is essential for the development of non-small cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Janus Quinase 2/metabolismo , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3 , Proteínas de Fusão Oncogênica/genética , Transdução de SinaisRESUMO
Ovarian cancer is prone to recurrence and chemotherapy resistance. Ovarian tumours of some patients have been positive for anaplastic lymphoma kinase fusion gene expression (ALK+). Preclinical studies indicate that anaplastic lymphoma kinase inhibitor can suppress the growth of ovarian cancer cells and transplantation tumours. Here, we present a patient with metastatic ALK+ high-grade serous ovarian cancer that testing positive for EML4-ALK (microtubule-associated protein-like 4 gene, fused to the anaplastic lymphoma kinase gene), experienced dramatic benefit after administration of the anaplastic lymphoma kinase inhibitor alectinib. This is the first clinical evidence that treatment with alectinib may provide a personalized maximum benefit for patients with high-grade serous ovarian cancer who are positive for EML4-ALK.
Assuntos
Carbazóis/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Neoplasias Ovarianas/tratamento farmacológico , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinase do Linfoma Anaplásico/genética , Carcinoma Epitelial do Ovário/genética , Feminino , Humanos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Ovarianas/genética , Resultado do TratamentoRESUMO
INTRODUCTION: Ceritinib is a selective second-generation ALK inhibitor that is highly sensitive to echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) molecule. CASE REPORT: In this paper, we report a 68-year-old female that was diagnosed with stage 4 ALK-positive non-small cell lung cancer (NSCLC).Management and outcome: She was treated with crizotinib first-line, cisplatin and gemcitabine as second-line. And for third-line, ceritinib was started. She had complete response over 3.5 years under ceritinib treatment. And she is still receiving ceritinib with no adverse event. DISCUSSION: Cases achieving complete response with ceritinib treatment are rare. In this paper, we aimed to emphasize the complete response in stage 4 NSCLC in an elderly patient.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Sulfonas/uso terapêutico , Idoso , Feminino , HumanosRESUMO
BACKGROUND: Several genetic driver alterations have been identified in micropapillary lung adenocarcinoma (MPA). However, the frequency of co-alteration of ROS1, EGFR, and EML4-ALK is yet unclear. Herein, we investigated the relationship between clinicopathologic characteristics and well-identified driver mutations of MPA compared with non-micropapillary lung adenocarcinoma (LA). METHODS: Formalin-fixed paraffin-embedded (FFPE) sections derived from lung adenocarcinoma patients who never received adjuvant chemotherapy or radiation therapy prior to surgical resection were collected from October 2016 to June 2019. EGFR mutations, ROS1 rearrangements, and EML4-ALK fusion were identified in a set of 131 MPA and LA cases by using the amplification refractory mutation system (ARMS). The response rate and duration of response were assessed using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). RESULTS: EGFR mutations had occurred in 42 (76.4%) MPA patients and 42 (55.3%) LA patients. Interestingly, ROS1 rearrangements were highly enriched only in the MPA cases (6/55, 10.9%) but rarely in the LA cases (1/76, 1.3%). Furthermore, 7.3% (4/55) MPA samples had double gene mutations, while only 1.3% (1/76) LA cases had double gene alterations. Of 5 patients with harboring two driver oncogene mutations, four patients (80%) obtained partial response, and one patient (20%) suffered recurrence. CONCLUSIONS: A higher prevalence of ROS1 rearrangement or combined mutations of ROS1, EGFR, and EML4-ALK may play a critical role in the tumorigenesis of MPA. These findings provide a novel therapeutic strategy for patients with malignant MPA through combining TKIs than one TKI.
Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Adenocarcinoma/genética , Adenocarcinoma de Pulmão/genética , China , Humanos , Neoplasias Pulmonares/genética , Mutação , Recidiva Local de Neoplasia , Proteínas de Fusão Oncogênica/genética , Patologia Molecular , Prognóstico , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/genéticaRESUMO
RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA-guide RNA complex residing at the 5' of the crRNA. The activity of Cas13a was maximal for guide RNAs 24-30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70-76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.
Assuntos
Sistemas CRISPR-Cas/genética , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Proteínas de Fusão Oncogênica/genética , Caspases/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , RNA/genética , Interferência de RNARESUMO
Tyrosine kinase inhibitors (TKI) have improved prognosis in metastatic anaplastic lymphoma kinase (ALK)-driven lung adenocarcinoma, but patient outcomes vary widely. We retrospectively analyzed the clinical course of all cases with assessable baseline TP53 status and/or ALK fusion variant treated at our institutions (n = 102). TP53 mutations were present in 17/87 (20%) and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK variant 3 (V3) in 41/92 (45%) patients. The number of metastatic sites at diagnosis was affected more by the presence of V3 than by TP53 mutations, and highest with both factors (mean 5.3, p < 0.001). Under treatment with ALK TKI, progression-free survival (PFS) was shorter with either TP53 mutations or V3, while double positive cases appeared to have an even higher risk (hazard ratio [HR] = 2.9, p = 0.015). The negative effect of V3 on PFS of TKI-treated patients was strong already in the first line (HR = 2.5, p = 0.037) and decreased subsequently, whereas a trend for PFS impairment under first-line TKI by TP53 mutations became stronger and statistically significant only when considering all treatment lines together. Overall survival was impaired more by TP53 mutations (HR = 4.9, p = 0.003) than by V3 (HR = 2.4, p = 0.018), while patients with TP53 mutated V3-driven tumors carried the highest risk of death (HR = 9.1, p = 0.02). Thus, TP53 mutations and V3 are independently associated with enhanced metastatic spread, shorter TKI responses and inferior overall survival in ALK+ lung adenocarcinoma. Both markers could assist selection of cases for more aggressive management and guide development of novel therapeutic strategies. In combination, they define a patient subset with very poor outcome.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação , Proteínas de Fusão Oncogênica/genética , Proteína Supressora de Tumor p53/genética , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Proteína Supressora de Tumor p53/metabolismoRESUMO
With the advent of next-generation sequencing (NGS) and precision medicine, investigators have determined that tumors from different tissue sources that have the same types of genetic mutations will have a positive response to the same targeted therapy. This finding has prompted us to seek potential therapeutic targets for patients with carcinoma of unknown primary (CUP) using NGS technology. Here, we reported a case of a woman with CUP resistance to chemotherapy. We detected 450 cancer-related gene alterations using three metastatic tumor specimens and found the presence of EML4 exon13 and ALK exon20 fusion. The tumor did respond to crizotinib, a first-generation ALK inhibitor. When her tumor progressed, circulating tumor DNA detection revealed ALK L1196 M and G1269A mutation resistance to crizotinib, but she had a response to brigatinib. This case revealed that NGS technology used to detect the genetic alterations in patients with CUP might be a reliable method to find potential therapeutic targets, although the primary lesion could not always be confirmed. KEY POINTS: This case exemplifies responsiveness to ALK inhibitor in carcinoma of unknown primary (CUP) with EML4-ALK fusion.Next-generation sequencing is an important diagnostic tool to find potential therapeutic targets in CUP.Liquid biopsy may be useful to provide critical information about resistance mechanisms in CUP to guide sequential treatment decision with targeted therapy.