RESUMO
Toll-like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non-small-cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K-ras (K-ras(LA1) ), with and without TLR9 inactivation (K-ras(LA1) TLR9(-/-) and K-ras(LA1) TLR9(+/+) , respectively). TLR9 was functionally expressed only in mononuclear cells of K-ras(LA1) TLR9(+/+) mice. These mice had significantly worse survival and a higher tumor burden than K-ras(LA1) TLR9(-/-) mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio-Plex multiplex bead-based assays. Factor VIII was assessed by immunochemistry. Tumors from K-ras(LA1) TLR9(+/+) mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K-ras(LA1) TLR9(-/-) mice. LKR13 cells, an ADC cell line derived from K-ras(LA1) mice, were subcutaneously injected into TLR9(-/-) and TLR9(+/+) mice. Syngeneic tumors regressed in TLR9(-/-) but not in TLR9(+/+) mice. Peripheral blood mononuclear cells from TLR9(-/-) mice released less VEGF than those from TLR9(+/+) mice. In 61 patients with early-stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/mortalidade , Inflamação/imunologia , Leucócitos Mononucleares/patologia , Neoplasias Pulmonares/mortalidade , Neovascularização Patológica , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptor Toll-Like 9/fisiologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Carga Tumoral , Fator A de Crescimento do Endotélio VascularRESUMO
The appropriate selection of patients is a major challenge in the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Prospective trials in adenocarcinoma demonstrated that the mucinous subtype presents a poorer outcome under EGFR-TKI treatment than the non-mucinous subtype. Our aim was to determine the molecular characteristics associated with resistance to EGFR-TKIs in mucinous and non-mucinous adenocarcinoma. Eighty adenocarcinoma samples, including 34 tumours from patients treated with gefitinib in a phase II clinical trial (IFCT0401), were classified as mucinous (n = 32) or non-mucinous (n = 48) adenocarcinoma. We demonstrated that four biological markers were differentially expressed between the two subtypes: mucinous tumours that overexpressed IGF1R (p < 0.0001) and amphiregulin (p = 0.004) with a tendency for more frequent KRAS mutations, in contrast to non-mucinous tumours that overexpressed EGFR (p < 0.0001) and TTF-1 (p < 0.0001) with more frequent EGFR mutations (p = 0.037). Higher IGF1R (p = 0.02) and lower TTF-1 (p = 0.02) expression was associated with disease progression under gefitinib treatment. We observed in vitro cross-talk between EGFR and IGF1R signalling pathways in gefitinib-resistant H358 mucinous cells. Anti-amphiregulin siRNAs and anti-IGF1R treatments sensitized the H358 cells to gefitinib-induced apoptosis with additive effects, suggesting that these treatments could overcome the resistance of mucinous tumours to EGFR-TKIs, including those with KRAS mutation. Our results highlighted that mucinous and non-mucinous adenocarcinoma subtypes are different entities with different therapeutic responses to EGFR-TKIs. These data will foster the development of therapeutic strategies for treating adenocarcinoma with mucinous component.
Assuntos
Adenocarcinoma Mucinoso/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anfirregulina , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Família de Proteínas EGF , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/metabolismo , Quinazolinas/uso terapêutico , Fatores de Transcrição , Resultado do Tratamento , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
INTRODUCTION: HIV is an exclusion criterion for most lung cancer (LC) trials, however LC is the most common non-AIDS-defined malignancy in people living with HIV (PLHIV), poorer prognosis than the general population. Circulating tumor DNA (ctDNA) was a prognostic marker in LC patients from the general population. This study assessed ctDNA's prognostic value in PLHIV from a dedicated phase II trial. METHODS: Overall, 61 PLHIV with advanced non-squamous non-small-cell lung cancer (NSCLC) participated in the IFCT Phase II trial evaluating first-line four-cycle carboplatin (Ca) AUC5 pemetrexed (P) 500 mg/m2 induction therapy every 3 weeks, followed by P maintenance therapy. Blood samples collected before treatment were analyzed to detect ctDNA using ultra-deep targeted next-generation-sequencing (NGS). RESULTS: Appropriate samples were available from 55 PLVIH and analyzed for ctDNA detection. Including 42 males (76.4 %), 52.9 years median age, 51 smokers (92.7 %), five with non-squamous NSCLC Stage III (9%), 50 Stage IV (91 %), and performance status (PS) 0-2. ctDNA was detected in 35 patients (64 %), 22 with high and 13 with low ctDNA levels. Overall, 77 % were positive for TP53, 29 % for KRAS, and 11 % for STK11 mutations, more than one alteration was detected in 43 % of samples. Multivariate analysis showed that positive ctDNA was significantly associated with shorter PFS (HR, 4.31, 95 %CI: 2.06-8.99, p < 0.0001), and shorter OS (HR, 3.52, 95 %CI: 1.72-7.19, p < 0.001). Moreover, OS was significantly longer for patients with low ctDNA levels at diagnosis as compared to high (p = 0.01). CONCLUSION: We show that ctDNA detection using ultra-deep NGS is an independent prognostic factor in PLHIV with advanced NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , DNA Tumoral Circulante , Infecções por HIV , Neoplasias Pulmonares , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
INTRODUCTION: Immune checkpoint inhibitors (ICIs) have improved cancer prognosis but have not been evaluated specifically in sarcomatoid carcinoma (SC), a rare lung cancer subtype with poor prognosis. As such, our study sought to retrospectively assess the efficacy of ICI in SC. METHODS: All consecutive patients with centrally confirmed SC treated using ICI as a second-line treatment or beyond between 2011 and 2017 were enrolled. Programmed death-ligand 1 (PD-L1) tumor expression was assessed using immunohistochemistry (SP263 clone) and the tumor mutational burden (TMB) with the Foundation One panel. TMB was considered high if it was greater than or equal to 10 mutations per megabase. RESULTS: Overall, 37 patients with SC were evaluated, predominantly men (73%) with a median age of 63.2 years (36.8-79.7) and who were current or former smokers (94.6%). Immunotherapy (nivolumab, 86.5% of cases) was given as a second-line treatment in 54% of the patients and as third-line treatment or beyond in 46% of the patients. The objective response rate was 40.5% and disease control rate was 64.8%, regardless of PD-L1 status. Median overall survival was 12.7 months (range: 0.3-45.7). One-third of patients exhibited early progression. The median PD-L1 expression was 70% (0-100). There was a trend toward higher PD-L1 expression in responsive diseases, with an objective response rate of 58.8% in patients with PD-L1+ and 0% in the one patient with PD-L1- (p = 0.44). The median TMB was 18 (4-39) mutations per megabase, with 87.5% of the cases displaying a high TMB. There was a trend toward higher TMB in responders versus stable or progressive diseases (p = 0.2). CONCLUSIONS: Patients with SC exhibited high response rates and prolonged overall survival under ICI treatment. These data support the prospective investigation of ICI in patients with SC who are under first-line treatment.
Assuntos
Carcinoma , Neoplasias Pulmonares , Antígeno B7-H1/uso terapêutico , Humanos , Inibidores de Checkpoint Imunológico , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.
Assuntos
Adenocarcinoma/metabolismo , Líquido da Lavagem Broncoalveolar/química , Calpaína/análise , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor 2 Toll-Like/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Calpaína/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Inflamação/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias/análise , Neutrófilos/metabolismo , PrognósticoRESUMO
BACKGROUND: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have shown efficacy in the treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), but the disease eventually progresses in all patients. In many cases, resistance to ALK TKIs arises through ALK mutations. Although clinical and biological data suggest variations in TKI efficacy according to the mechanism of resistance, ALK mutations are still rarely investigated in routine practice. MATERIALS AND METHODS: We performed a retrospective multicentric study with an aim to determine the frequency and clinical relevance of ALK alterations detected using targeted next-generation sequencing in patients with advanced ALK-rearranged NSCLC after progression during an ALK TKI treatment. Data on clinical, pathological, and molecular characteristics and patient outcomes were collected. RESULTS: We identified 23 patients with advanced ALK-rearranged NSCLC who, between January 2012 and May 2017, had undergone at least 1 repeat biopsy at progression during an ALK TKI treatment. A resistance mechanism was identified in 9 of the 23 patients (39%). The anomalies involved included 9 ALK mutations in 8 patients and one ALK amplification. The ALK mutation rate was 15% after failure of a first ALK TKI and 33% after failure of 2 ALK TKI treatments. Five of 7 patients who received a different ALK TKI after detection of an ALK mutation achieved an objective response. All of the patients who received a TKI presumed to act on the detected ALK mutant achieved disease control. CONCLUSION: Targeted next-generation sequencing is suitable for detecting ALK resistance mutations in ALK-rearranged NSCLC patients in routine practice. It might help select the best treatment at the time of disease progression during treatment with an ALK TKI.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Crizotinibe/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Adolescente , Adulto , Idoso , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Rearranjo Gênico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos Retrospectivos , Adulto JovemRESUMO
PURPOSE: Adenocarcinoma with bronchioloalveolar carcinoma (BAC) features is a subtype of non-small cell lung cancers characterized by an intense inflammatory reaction composed of macrophages and neutrophils and by a distinct natural history with intrapulmonary spread leading to death due to respiratory failure. We hypothesized that neutrophils could promote aerogenous spread of lung adenocarcinoma with BAC features. EXPERIMENTAL DESIGN: We examined the effect of neutrophils on A549 cell line detachment in vitro and we quantified desquamation of tumor cells on tumor tissue (n = 25) and on matched bronchioloalveolar lavage (n = 17) in vivo in a series of patients with adenocarcinoma with BAC features. RESULTS: Neutrophils induced A549 detachment mediated by signals through cell-to-cell contact. Detached A549 cells were still viable and able to proliferate in vitro. Neutralization studies identified several membrane-bound molecules involved in detachment (i.e., intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, tumor necrosis factor alpha/tumor necrosis factor alpha receptor inhibitor, interleukin-1alpha /interleukin-1alpha receptor, and neutrophil elastase). In tumor tissue, shedding was detected in all samples, with a median shedding score of 42% (range, 4-95%). Micropapillary clusters were detected in 23 of the 25 tumor tissue samples, with a median micropapillary score of 1.40 (range, 0-2.1), and tumor cells were detected in 7 of 17 lavages. The micropapillary score was associated with a high neutrophil count in bronchioloalveolar lavage (P = 0.051). The shedding cell percentage was a significant factor in shorter survival (P = 0.034, univariate Cox analysis). CONCLUSIONS: Tumor shedding is induced by neutrophils. It is a significant factor of shorter survival and may be an important event in adenocarcinoma progression.
Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Adenocarcinoma/patologia , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/patologia , Neutrófilos/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma Bronquioloalveolar/metabolismo , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , MasculinoRESUMO
OBJECTIVE: Immunotherapies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) checkpoint improved prognosis in lung cancer. PD-1/PD-L1 status, however, has not been investigated in human immunodeficiency virus (HIV)-positive patients. This study assessed PD-L1 status and tumor immune-cell infiltration in nonsmall cell lung cancer (NSCLC) in HIV patients. METHODS: Consecutive HIV patients treated between 1996 and 2014 were enrolled. PD-L1 tumor expression was assessed using immunohistochemistry with two antibodies (clones 5H1 and E1L3N), and tumor immune-cell infiltration with CD3, CD4, CD8, CD20, CD163, and MPO. PD-L1 expression and immune infiltration results were compared with those of 54 NSCLCs from unknown HIV status patients. RESULTS: Thirty-four HIV-positive patients were evaluated: predominantly men (88.2%) (median age: 51.1 years) presenting stage IV (38.2%) adenocarcinomas (76.5%). The median blood CD4 count was 480 cells/µL (86-1120) and 64% exhibited undetectable viral load. The PD-L1 score (percentage of positive cellsâ×âintensity) was higher in HIV-positive than HIV-undetermined patients with the E1L3N clone [median (range) 0 (0-150) versus 0 (0-26.7), Pâ=â0.047], yet not with the 5H1 clone [0 (0-120) versus 0 (0-26.7) Pâ=â0.07, respectively]. PD-L1 expression frequency did not differ between both cohorts (18.7 versus 9.3% using E1L3N and 10 versus 5.6% using 5H1 clone, respectively). There were significantly greater cytotoxic T-cell (Pâ<â0.001), B-lymphocyte (Pâ=â0.005), and activated macrophage (Pâ<â0.001) infiltrations in the HIV-positive patients, but no differences for CD4 T cells. CONCLUSION: Tumors in HIV-positive patients seem to express higher PD-L1 levels with increased immune infiltration, supporting their inclusion in clinical trials assessing immune checkpoint inhibitors.
Assuntos
Antígeno B7-H1/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Infecções por HIV/complicações , Leucócitos/química , Neoplasias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: MNNG HOS transforming gene (MET) abnormalities such as amplification and exon 14 mutations may be responsive to targeted therapies. They are prevalent in lung sarcomatoid carcinomas (LSCs) and must be diagnosed as efficiently as possible. Hypothetically, c-MET overexpression by immunohistochemistry (IHC) may prove effective as a screening test for MET abnormalities. METHODS: Tissue samples were obtained from consecutive patients with a resected LSC in four oncologic centers. IHC was performed using the SP44 antibody (Ventana, Tucson, Arizona) and evaluated using the MetMab score and H-score. Fluorescence in situ hybridization was applied with the dual color probe set from Zytovision (Clinisciences, Nanterre, France). True MET amplification was diagnosed when MET gene copy number was 5 or greater and the ratio between MET gene copy number and chromosome 7 number was greater than 2. All MET exon 14 alterations including those affecting splice sites occurring within splice donor and acceptor sites were detected in the routine molecular testing on genetic platforms. RESULTS: A total of 81 LSCs were included. Fourteen (17%) exhibited positive IHC using the MetMab score and 15 (18.5%) using the H-score. MET amplification was detected in six tumors (8.5%) and MET exon 14 mutation in five (6%). A weak positive correlation between IHC and fluorescence in situ hybridization was found (r = 0.27, p = 0.0001). IHC sensitivity for MET amplification was 50%, with a specificity of 83%, positive predictive value of 21.4%, and negative predictive value of 94.7%. IHC sensitivity for MET exon 14 mutations was 20%, with a specificity of 83%, positive predictive value of 7%, and negative predictive value of 94%. CONCLUSION: IHC is not a relevant screening tool for MET abnormalities in LSC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Éxons/genética , Amplificação de Genes , Neoplasias Pulmonares/diagnóstico , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Gigantes/diagnóstico , Carcinoma de Células Gigantes/genética , Carcinoma de Células Gigantes/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Estudos de Coortes , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Sarcoma/diagnóstico , Sarcoma/genética , Sarcoma/metabolismoRESUMO
Invasive mucinous adenocarcinoma (IMA) is a mucinous variant of lepidic predominant lung adenocarcinoma (LPA) and associated with a worse prognosis. We postulated that cytokine expression would enable us to differentiate IMA from LPA in terms of prognosis and acquisition of pro-tumoural capacities. A 30-cytokine panel was assessed in bronchoalveolar lavage fluids (BALF) from IMA (n=38), LPA (n=25) and control samples (n=7). We investigated the expression of differentially expressed cytokines and splice variants of their receptors in surgical samples. The presence of EGFR and KRAS mutations were determined. We also examined the expression of cytokines and splice variants of their receptors in different cell lines, exploring their functional impact on signalling pathways, proliferation and migration. Only C-X-C motif chemokine 10 (CXCL10) was differentially expressed, namely overexpressed in IMA BALF compared with LPA. CXCL10 overexpression in BALF was linked to a worse prognosis. In surgical samples, CXCL10 and its receptor C-X-C motif chemokine receptor 3 (CXCR3) were overexpressed in IMA compared to LPA. A pro-tumoural CXCR3-A splice variant was overexpressed in IMA, suggesting a CXCL10/CXCR3-A autocrine loop in IMA. CXCL10 and CXCR3 expression were not correlated with EGFR or KRAS status. CXCL10 up-regulated CXCR3-A expression, Erk1/2 phosphorylation and enhanced migration in the mucinous H2228 cell line. CXCL10/CXCR3-A may play a pro-tumoural role in IMA via an autocrine mechanism.
RESUMO
OBJECTIVES: To evaluate MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression in invasive lepidic predominant adenocarcinoma (LPA) and invasive mucinous adenocarcinoma (IMA) of the lung, and the impact of oncogenic drivers. MATERIALS AND METHODS: MUC1, MUC2, MUC5B, MUC5AC, MUC6, TTF1 and Hnf4α immunohistochemistry was performed on surgical samples from 52 patients with IMA (n=25) or LPA (n=27). We searched for EGFR, KRAS, BRAF, and HER2 mutations and ALK, ROS1, and NRG1 rearrangements. RESULTS: MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression was detected in tumor cells in 77%, 2%, 63%, 36%, and 21% of cases, respectively. MUC1 was significantly more overexpressed in LPA. MUC5B, MUC5AC, and MUC6 were typically detected in goblet cells and overexpressed in IMA. Hnf4α-positive IMA (n=11) were TTF1-negative and typically did not expressed MUC1 and expressed MUC5AC and MUC6. Hnf4α-negative IMA (n=14) showed a reverse profile of mucins expression, with MUC1 expression and a lack of MUC5AC and MUC6 expression. EGFR-positive status was significantly associated with LPA, MUC1 expression, and no MUC5B, MUC5AC, or MUC6 expression. KRAS-positive status was significantly associated with IMA and MUC5B and MUC5AC expression. CONCLUSIONS: LPA and IMA exhibit specific mucin expression profiles, with MUC1 being associated with LPA, while MUC5B, MUC5AC, and MUC6 were associated with IMA. Hnf4α expression and EGFR and KRAS mutations may play a role in mucin expression profiles of these lung adenocarcinoma subtypes.
Assuntos
Adenocarcinoma Mucinoso/metabolismo , Epitélio/fisiologia , Neoplasias Pulmonares/metabolismo , Mucinas/metabolismo , Alvéolos Pulmonares/patologia , Adenocarcinoma Mucinoso/genética , Idoso , Biomarcadores Tumorais/genética , Carcinogênese/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes erbB-1/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/genética , Masculino , Mucinas/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Increased numbers of tumor-infiltrating neutrophils are linked to poorer outcome in patients with adenocarcinoma of the bronchioloalveolar carcinoma (BAC) subtype. Hepatocyte growth factor (HGF) is a pleiotropic cytokine operating through activation of the proto-oncogene c-met and is a factor of poor prognosis in various cancers. Reports that neutrophils produce HGF led us to investigate their participation in the aerogenous spread of tumor cells and the prognosis of BAC, through the effect of HGF on c-met-expressing tumor cells. Immunoreactive HGF was detected in bronchoalveolar lavage fluid (BALF) supernatants from 34 of 36 patients, whereas it was undetectable in BALF from healthy controls. The HGF thus detected was locally produced, because HGF mRNA was expressed by the patients' fresh alveolar cells, and HGF protein was detected in 24-h culture supernatants. In immunocytochemical studies of BALF cytospin preparations and tumor specimens from the patients, neutrophils were always HGF-positive, whereas alveolar macrophages and tumor cells gave inconsistent results. Alveolar neutrophil-derived HGFs induced significant, concentration-dependent migration of BAC-derived tumor cells in vitro, and this effect was inhibited by anti-HGF neutralizing antibodies. Granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha (present in the lung tumor microenvironment) provoked HGF release from neutrophil intracellular stocks, and the capacity of blood neutrophils from BAC patients to produce HGF was unaltered. Immunochemical studies of c-met expression in BALF cytospin preparations and tumor sections showed that most HGF receptor-bearing cells were tumor cells. High HGF levels in BALF supernatants were significantly associated with poorer outcome in patients with BAC and were an independent predictor of clinical outcome in multivariate analysis. Altogether, our results support the notion that BAC generates a local environment that attracts functionally normal neutrophils from peripheral blood and leads to neutrophil release of biologically active HGF on contact with HGF receptor-expressing tumor cells, thereby contributing to poorer patient outcome.
Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Fator de Crescimento de Hepatócito/biossíntese , Neoplasias Pulmonares/patologia , Neutrófilos/metabolismo , Adenocarcinoma Bronquioloalveolar/imunologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Progressão da Doença , Feminino , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neutrófilos/imunologia , Prognóstico , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/biossínteseRESUMO
Invasive mucinous lung adenocarcinoma (IMA) is a rare subtype of lung adenocarcinoma with no effective treatment option in advanced disease. KRAS mutations occur in 28-87% of the cases. NRG1 fusions were recently discovered in KRAS-negative IMA cases and otherwise negative for known driver oncogenes and could represent an attractive therapeutic target. Published data suggest that NRG1 fusions occur essentially in nonsmoking Asian women. From an IMA cohort of 25 French patients of known ethnicity, driver oncogenes EGFR, KRAS, BRAF, ERBB2 mutations, and ALK and ROS1 rearrangements presence were analyzed. In the IMA samples remaining negative for these driver oncogenes, an NRG1 rearrangement detection was performed by FISH. A driver oncogene was identified in 14/25 IMA, namely 12 KRAS mutations (48%), one ROS1 rearrangement (4%), and one ALK rearrangement (4%). The detection of NRG1 rearrangement by FISH was conducted in the 11 pan-negative IMA. One sample was NRG1FISH-positive and 100% of the tumor nuclei analyzed were positive. This NRG1-positive patient was a 61-year-old nonsmoking woman of Vietnamese ethnicity and was the sole patient of Asian ethnicity of the cohort. She died 6 months after the diagnosis with a pulmonary multifocal disease. NRG1FISH detection should be considered in patients with IMA pan-negative for known driver oncogenes. These results might suggest that NRG1 fusion is more frequent in IMA from Asian patient. Larger studies are needed.
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Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neuregulina-1/genética , Proteínas de Fusão Oncogênica/genética , Adenocarcinoma de Pulmão , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Neuregulina-1/metabolismo , Proteínas de Fusão Oncogênica/metabolismoRESUMO
INTRODUCTION: Chemoresistance is a major challenge in the treatment of advanced non-small-cell lung cancer (NSCLC). Because the Sonic hedgehog (Shh) pathway is reactivated in NSCLC, we investigated an association between chemoresistance and Shh activation. PATIENTS AND METHODS: From a cohort of 178 patients with advanced NSCLC treated with platinum-based chemotherapy as first-line treatment, we selected all surgical tumor samples at diagnosis (n = 36). Shh activation was evaluated through Gli1 and Gli2 expression using immunohistochemistry (quantitative score). In vitro treatment studies with cisplatin or vismodegib (Shh pathway inhibitor), or both, were performed on NSCLC cell lines (H322 and A549) and primary cultures from patients with sarcomatoid carcinoma (n = 4). RESULTS: Of the 36 patients, 12 had NSCLC refractory to chemotherapy (R-patients, 33.3%) and 24 had controlled disease (C-patients). Gli1 expression did not differ between the R- and C-patients (P = .35). Gli2 expression was more often positive in the R-patients (41.7% vs. 8.3%; P = .02). Progression-free survival (PFS) and overall survival (OS) in patients with a Gli2-positive score was 2.1 and 8.0 months, respectively, compared with 6.7 and 18.0 months for patients with a Gli2-negative score (P = .03 and P = .002, respectively). On multivariate analysis, the Gli2 score correlated independently with PFS (hazard ratio [HR], 2.64; 95% confidence interval [CI], 1.05-6.63; P = .04) and OS (HR, 4.36; 95% CI, 1.67-11.36; P = .003). The sarcomatoid carcinoma cell lines were more resistant to cisplatin than were the H838 and A549 cell lines. The cisplatin-vismodegib combination displayed a synergistic cytotoxic effect in the most chemoresistant cells in vitro. CONCLUSION: The Shh pathway is associated with resistance to platinum-based chemotherapy in NSCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Compostos de Platina/uso terapêutico , Adulto , Anilidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Ativação Transcricional/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
OBJECTIVES: Pulmonary sarcomatoid carcinomas (SC) are rare tumors, associated with worse prognosis and resistant to platinum-based regimens. Therapies targeting the PD-1/PD-L1 pathway are an emerging treatment for lung cancer. By characterizing intra-tumoral immune infiltration and evaluating PD-L1 expression, it could be possible to predict the efficacy of these new treatments. MATERIALS AND METHODS: From 1997 to 2013, data from all patients with SC who underwent lung resection was collected. Tumor-immune infiltration and PD-L1 expression were studied by immunochemistry tests, analyzing CD3 (clone SP7), CD4 (clone 1F6), CD8 (clone C8/144b), CD20 (clone L26), CD163 (clone 10D6), MPO (clone 59A5), and PD-L1 (clone 5H1). Results were compared to those of 54 NSCLC. RESULTS: In total, 75 SC were included. Forty (53%) SC expressed PD-L1 vs 11 NSCLC (20%) (p<0.0001). CD3+ tumor-infiltrating lymphocytes and CD163+ tumor-associated macrophages were more important in SC than in NSCLC (median 23% [17-30] of tumoral surface vs 17% [7-27], p=0.011 and 23% [17-30] vs 20% [13-23], p=0.002, respectively). In SC, the presence of Kirsten Ras (KRAS) mutations, blood vessel invasion, and TTF1+ positivity were associated with PDL1 expression. On multivariate analysis, only CD163+ macrophages and blood-vessel invasion were associated with tumoral PD-L1 expression. High levels of tumor-infiltrating lymphocytes (CD3+ or CD4+ and not CD8+) constituted a factor of good prognosis on survival. Interestingly, PD-L1 expression distinguishes subpopulations within tumor-infiltrating lymphocytes (CD3+ or CD4+) with different prognosis CONCLUSIONS: PD-L1 expression was higher in SC than in NSCLC as well as immune-cell infiltration by TCD3 cells and macrophages. This suggests that targeting the PD-1/PD-L1 pathway could represent a new potential therapy.
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Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Adulto , Idoso , Antígeno B7-H1/genética , Biomarcadores Tumorais , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Razão de Chances , PrognósticoRESUMO
PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.
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Análise Mutacional de DNA/métodos , Genes p53 , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Oligonucleotídeos/genética , Idoso , Alelos , Biópsia , Estudos de Coortes , DNA/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estudos Prospectivos , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVES: This study investigated the clinical and prognostic impact of intracytoplasmic mucin in lung adenocarcinoma with "pneumonic" radiological presentation, formerly known as bronchioloalveolar carcinoma (BAC). PATIENTS AND METHODS: Between 1986 and 2011, clinical and pathological data from 120 consecutive patients with lung adenocarcinoma with "pneumonic" radiological presentation were reviewed. Intracytoplasmic mucin was assessed using a diastase-resistant periodic acid-Schiff staining. The presence of EGFR or K-Ras mutations and ALK rearrangement were determined in surgical samples. RESULTS: The two predominant histological patterns were invasive mucinous adenocarcinoma (40%) and lepidic predominant adenocarcinoma (32%). Intracytoplasmic mucin was detected in 71 patients (59.2%) who were more likely to be non-smokers (p=0.04) and have bronchorrhea (p=0.006), crepitant rales (p=0.02), or neutrophil alveolitis (p=0.0004). In mucin-producing tumors, EGFR mutation was not detected, K-Ras mutations and ALK rearrangement were present in 32% and 3% of cases, respectively. In non-mucin-producing tumors, EGFR and K-Ras mutations were detected in 17% and 10% of cases, respectively, no ALK rearrangement was detected. In univariate analysis, performance status>0, crepitant rales, bronchorrhea, neutrophil alveolitis, bilateral extension, intracytoplasmic mucin and no surgery were associated with worse survival. In multivariate analyses, intracytoplasmic mucin, neutrophil alveolitis, and no surgery were independent factors for worse survival. CONCLUSION: Intracytoplasmic mucin is associated with specific clinical characteristics and is an independent factor for worse survival in lung adenocarcinoma formerly known as BAC.
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Adenocarcinoma Bronquioloalveolar/diagnóstico , Adenocarcinoma Mucinoso/diagnóstico , Citoplasma/metabolismo , Neoplasias Pulmonares/diagnóstico , Mucinas/metabolismo , Neutrófilos/imunologia , Adenocarcinoma Bronquioloalveolar/mortalidade , Quinase do Linfoma Anaplásico , Receptores ErbB/genética , Feminino , Genes ras/genética , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Reação do Ácido Periódico de Schiff , Prognóstico , Receptores Proteína Tirosina Quinases/genética , Fatores de Risco , Análise de SobrevidaRESUMO
Epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR-TKI) are a therapeutic option as second-line therapy in non-small-cell lung carcinoma (NSCLC), regardless of the EGFR gene status. Identifying patients with early progression during EGFR-TKI treatment will help clinicians to choose the best regimen, TKI or chemotherapy. From a prospective database, all patients treated with gefitinib or erlotinib between 2001 and 2010 were retrospectively reviewed. Patients were classified into two groups according to their tumor response by RECIST after 45 days of treatment, progressive disease (PD) or controlled disease (CD). Two hundred and sixty-eight patients were treated with EGFR-TKI, among whom 239 were classified as PD (n = 75) and CD (n = 164). Median overall survival was 77 days (95% CI 61-109) for PD and 385 days (95% CI 267-481) for CD. Patients with PD were of younger age (P = 0.004) and more frequently current smokers (P = 0.001) had more frequently a performance status ≥2 (P = 0.012), a weight loss ≥10% (P = 0.025), a shorter time since diagnosis (P < 0.0001), a pathological classification as non-otherwise-specified NSCLC (P = 0.01), and the presence of abdominal metastases (P = 0.008). In multivariate analysis, abdominal metastases were the only factor associated with early progression (odds ratio (OR) 2.17, 95% CI [1.12-4.19]; P = 0.021). Wild-type EGFR versus mutated EGFR was associated with early progression. The presence of abdominal metastasis was independently associated with early progression in metastatic NSCLC receiving EGFR-TKI.
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Carcinogênese/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Inibidores de Proteínas Quinases/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Progressão da Doença , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Feminino , Gefitinibe , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Quinazolinas/administração & dosagemRESUMO
K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.
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Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Aerossóis , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Área Sob a Curva , Disponibilidade Biológica , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Imuno-Histoquímica , Injeções Intraperitoneais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neovascularização Patológica/genética , Neovascularização Patológica/prevenção & controle , Proteínas Proto-Oncogênicas p21(ras)/genética , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVES: Pulmonary sarcomatoid carcinomas (SC) are highly disseminated types of non-small-cell lung carcinoma. Their prognosis is poor. New therapeutic targets are needed to improve disease management. MATERIALS AND METHODS: From 1995 to 2013, clinical and survival data from all consecutive patients with surgically treated SC were collected. Pathological and biomarker analyses were performed: TTF1, P63, c-MET and ALK expression (immunohistochemistry), PAS staining, ALK rearrangement (FISH), and EGFR, KRAS, HER2, BRAF, PIK3CA, and MET genes mutations (PCR). RESULTS: Seventy-seven patients were included. Median age was 61 years (53-69). Histological subtypes were pleomorphic carcinoma (78%), carcinosarcoma (12%), and giant-cell and/or spindle-cell carcinoma (10%). Blood vessel invasion (BVI) was present in 90% of cases. Morphology and immunohistochemistry were indicative of an adenocarcinoma, squamous, and adenosquamous origin in 41.5%, 17% and 11.5%, respectively, 30% remained not-otherwise-specified. KRAS, PIK3CA, EGFR, and MET mutations were found in 31%, 8%, 3%, and 3%, respectively. No tumors had HER2 or BRAF mutations, or ALK rearrangement, whereas 34% had a c-MET positive score. Five-year overall survival (OS) was 29% for the whole population. At multivariate analysis, tumor size <50mm (HR=1.96 [1.04-3.73], p=0.011), no lymph-node metastasis (HR=3.25 [1.68-6.31], p<0.0001), no parietal pleural invasion (HR=1.16 [1.06-1.28], p=0.002), no BVI (HR=1.22 [1.06-1.40], p=0.005), and no squamous component (HR=3.17 [1.48-6.79], p=0.01) were associated with longer OS. Biomarkers did not influence OS. CONCLUSION: Dedifferentiation in NSCLC could lead to SC and an epithelial subtype component could influence outcome. BVI was present in almost all SCs and was an independent factor of poor prognosis.