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1.
Front Oncol ; 12: 999626, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36249060

RESUMO

ARID1A is frequently mutated in colorectal cancer (CRC) cells. Loss of ARID1A function compromises DNA damage repair and increases the reliance of tumor cells on ATR-dependent DNA repair pathways. Here, we investigated the effect of ionizing radiation (IR), in combination with ATR inhibitors (ATRi) in CRC cell lines with proficient and deficient ARID1A. The concept of selective vulnerability of ARID1A deficient CRC cells to ATRi was further tested in an ex vivo system by using the ATP-tumor chemosensitivity assay (ATP-TCA) in cells from untreated CRC patients, with and without ARID1A expression. We found selective sensitization upon ATRi treatment as well as after combined treatment with IR (P<0.001), especially in ARID1A deficient CRC cells (P <0.01). Knock-down of ARID1B further increased the selective radiosensitivity effect of ATRi in ARID1A negative cells (P<0.01). Mechanistically, ATRi abrogates the G2 checkpoint (P<0.01) and homologous recombination repair (P<0.01) in ARID1A deficient cells. Most importantly, ex-vivo experiments showed that ATRi had the highest radiosensitizing effect in ARID1A negative cells from CRC patients. Collectively, our results generate pre-clinical and clinical mechanistic rationale for assessing ARID1A defects as a biomarker for ATR inhibitor response as a single agent, or in a synthetic lethal approach in combination with IR.

2.
Cancers (Basel) ; 14(13)2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35804869

RESUMO

Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite local tumor control, no effective therapy has been found to prevent metastasis, resulting in a high mortality rate. In the present study, we evaluated the anti-tumor potential of non-selective ß-blockers in 3D tumor spheroids grown from UM cell lines. Of the various ß-blockers tested, carvedilol and its enantiomers were most potent in decreasing the viability of Mel270 spheroids. Carvedilol at a concentration of 10-50 µM significantly elicited cytotoxicity and induced apoptosis in spheroid cells. In result, carvedilol inhibited tumor spheroid growth and compactness, and furthermore prevented the long-term survival and repopulation of spreading spheroid cells. The drug sensitivity of the different spheroids grown from Mel270, 92-1, UPMD2, or UPMM3 cell lines was dependent on 3D morphology rather than on high-risk cytogenetic profile or adrenergic receptor expression levels. In fact, the monosomy-3-containing UPMM3 cell line was most responsive to carvedilol treatment compared to the other cell lines. The concurrent treatment of UPMM3 spheroids with carvedilol and 5 or 10 Gy irradiation revealed additive cytotoxic effects that provided tumor control. Collectively, our data demonstrate the anti-tumor properties of carvedilol and its enantiomers, which may serve as candidates for the co-adjuvant therapy of UM.

3.
Cell Death Dis ; 13(6): 529, 2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35668077

RESUMO

Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis.


Assuntos
Neoplasias Pulmonares , Recidiva Local de Neoplasia , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Proteínas Qa-SNARE/genética , Tolerância a Radiação/genética
4.
Cancer Control ; 28: 10732748211033497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34554888

RESUMO

PURPOSE: To assess the relative efficacy and safety of first-line systemic therapies in patients with metastatic colorectal cancer. EXPERIMENTAL DESIGN: A comprehensive literature review was conducted including MEDLINE, Embase, and the Cochrane Central Registry of Controlled Trials for phase II or III randomized controlled trials (RCTs) published up to and including July 15, 2019. We included RCTs in which at least 1 intervention was either chemotherapeutic agents (such as fluorouracil, irinotecan, or oxaliplatin) or antibodies targeting angiogenesis (such as bevacizumab) or agents that act on the epidermal growth factor receptor pathway (such as cetuximab and panitumumab) or studies reported at least one of the following outcomes: overall survival (OS), progression-free survival (PFS), and/or Grade 3 + adverse events (AEs). Using a random effect model, we performed a Bayesian network meta-analysis to analyze the probability of optimal therapeutic regime obtained from direct comparisons with indirect evidences. We estimated hazard ratios for OS and PFS. RESULTS: A total of 30 RCTs comprising 12,146 mCRC patients with 25 different treatment strategies were included. The triple combination FOLFOXIRI [fluorouracil, leucovorin, oxaliplatin, and irinotecan] plus bevacizumab provided significant survival benefits with improved OS over all other treatments. The network meta-analysis also indicated a significant advantage of using FOLFOXIRI plus bevacizumab in comparison to other treatment strategies for PFS. Besides, FOLFOXIRI plus bevacizumab was associated with the well-tolerated adverse events. CONCLUSIONS: Our study supported the use of FOLFOXIRI plus bevacizumab as the best first-line regimen and potentially effective and safe strategy for the management of patients with mCRC.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Neoplasias Colorretais/mortalidade , Humanos , Bloqueio Interatrial , Metanálise em Rede , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sobrevida
5.
Cancers (Basel) ; 13(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205625

RESUMO

Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1, Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses.

6.
Pharmacogenomics J ; 21(1): 37-46, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32546699

RESUMO

Identifying patients with locally advanced head and neck carcinoma on high risk of recurrence after definitive concurrent radiochemotherapy is of key importance for the selection for consolidation therapy and for individualized treatment intensification. In this multicenter study we analyzed recurrence-associated single-nucleotide polymorphisms (SNPs) in DNA repair genes in tumor DNA from 132 patients with locally advanced head and neck carcinoma (LadHnSCC). Patients were treated with definitive radiotherapy and simultaneous cisplatin-based chemotherapy at six partner sites of the German Cancer Consortium (DKTK) Radiation Oncology Group from 2005 to 2011. For validation, a group of 20 patients was available. Score selection method using proportional hazard analysis and leave-one-out cross-validation were performed to identify markers associated with outcome. The SNPs rs1799793 and rs13181 were associated with survival and the same SNPs and in addition rs17655 with freedom from loco-regional relapse (ffLRR) in the trainings datasets from all patients. The homozygote major rs1799793 genotype at the ERCC2 gene was associated with better (Hazard ratio (HR): 0.418 (0.234-0.744), p = 0.003) and the homozygote minor rs13181 genotype at ERCC2 with worse survival (HR: 2.074, 95% CI (1.177-3.658), p = 0.017) in comparison to the other genotypes. At the ffLRR endpoint, rs1799793 and rs13181 had comparable prognostic value. The rs1799793 and rs13181 genotypes passed the leave-one-out cross-validation procedure and associated with survival and ffLRR in patients with LadHnSCC treated with definitive radiochemotherapy. While findings were confirmed in a small validation dataset, further validation is underway within a prospective biomarker study of the DKTK.


Assuntos
Cisplatino/administração & dosagem , Recidiva Local de Neoplasia/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Proteína Grupo D do Xeroderma Pigmentoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiorradioterapia , Cisplatino/efeitos adversos , Intervalo Livre de Doença , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/radioterapia , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia
8.
J Mol Diagn ; 22(6): 801-810, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32247864

RESUMO

This article compares the expression and applicability of biomarkers, from single genes and gene signatures, identified in patients with locally advanced head and neck squamous cell carcinoma using the GeneChip Human Transcriptome Array 2.0, nCounter, and real-time PCR analyses. Two multicenter, retrospective cohorts of patients with head and neck squamous cell carcinoma from the German Cancer Consortium Radiation Oncology Group who received postoperative radiochemotherapy or primary radiochemotherapy were considered. Real-time PCR was performed for a limited number of 38 genes of the cohort who received postoperative radiochemotherapy only. Correlations between the methods were evaluated by the Spearman rank correlation coefficient. Patients were stratified based on the expression of putative cancer stem cell markers, hypoxia-associated gene signatures, and a previously developed seven-gene signature. Locoregional tumor control was compared between these patient subgroups using log-rank tests. Gene expressions obtained from nCounter analyses were moderately correlated to GeneChip analyses (median ρ = approximately 0.68). A higher correlation was obtained between nCounter analyses and real-time PCR (median ρ = 0.84). Significant associations with locoregional tumor control were observed for most of the considered biomarkers evaluated by GeneChip and nCounter analyses. In general, all applied biomarkers (single genes and gene signatures) classified approximately 70% to 85% of the patients similarly. Overall, gene signatures seem to be more robust and had a better transferability among different measurement methods.


Assuntos
Quimiorradioterapia/métodos , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , Transcriptoma , Adulto Jovem
9.
Int J Radiat Biol ; 95(9): 1205-1219, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31287365

RESUMO

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status. Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2 Gy (SF2) assessed by clonogenic survival. Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1 h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24 h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4 Gy, but decreased both at higher doses of 20 Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines. Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular , Linhagem Celular Tumoral , Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
10.
Sci Rep ; 9(1): 1282, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718758

RESUMO

Cisplatin is an extensively used chemotherapeutic drug for lung cancer, but the development of resistance decreases its effectiveness in the treatments of non-small cell lung cancer (NSCLC). In this study, we examined the effects of metformin, a widely used antidiabetic drug, on cisplatin radiosensitization in NSCLC cell lines. Human NSCLC cell lines, A549 (cisplatin-resistant) and H460 (cisplatin-sensitive), were treated with metformin, cisplatin or a combination of both drugs before ionizing radiation. Cell proliferation, clonogenic assays, western blotting, cisplatin-DNA adduct formation and immunocytochemistry were used to characterize the treatments effects. Metformin increased the radiosensitivity of NSCLC cells. Metformin showed additive and over-additive effects in combination with cisplatin and the radiation response in the clonogenic assay in H460 and A549 cell lines (p = 0.018 for the interaction effect between cisplatin and metformin), respectively. At the molecular level, metformin led to a significant increase in cisplatin-DNA adduct formation compared with cisplatin alone (p < 0.01, ANOVA-F test). This was accompanied by a decreased expression of the excision repair cross-complementation 1 expression (ERCC1), a key enzyme in nucleotide excision repair pathway. Furthermore, compared with each treatment alone metformin in combination with cisplatin yielded the lowest level of radiation-induced Rad51 foci, an essential protein of homologous recombination repair. Ionizing radiation-induced γ-H2AX and 53BP1 foci persisted longer in both cell lines in the presence of metformin. Pharmacological inhibition of AMP-activated protein kinase (AMPK) demonstrated that metformin enhances the radiosensitizing effect of cisplatin through an AMPK-dependent pathway only in H460 but not in A549 cells. Our results suggest that metformin can enhance the effect of combined cisplatin and radiotherapy in NSCLC and can sensitize these cells to radiation that are not sensitized by cisplatin alone.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Metformina/farmacologia , Proteínas de Neoplasias/biossíntese , Tolerância a Radiação/efeitos dos fármacos , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia
11.
Mol Cancer Ther ; 18(3): 656-666, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30478150

RESUMO

Targeting of epigenetic regulators as the chromatin remodeler SWI/SNF is proving to be a promising therapeutic strategy for individualized treatment of cancer patients. Here, we tested whether targeting one of the two mutually exclusive subdomains of the SWI/SNF complex BRM/SMARCA2 can sensitize specifically non-small cell lung carcinoma (NSCLC) cells with mutations in the other subunit BRG1/SMARCA4 toward ionizing radiation (IR). Knockdown of BRM with siRNA or shRNA and its consequences for radiation sensitivity as measured by clonogenic survival and plaque-monolayer control was studied in different NSCLC lines with or without BRG1 mutations and in primary fibroblasts. Furthermore, the effect on double-strand break (DSB) repair markers measured by immunofluorescence staining of 53BP1-, γ-H2AX-, and Rad51-foci was investigated. BRG1-mutated cell lines showed an increased surviving fraction compared with BRG1 proficient cells. Depletion of BRM (i) leads to a decreased proliferation rate and plating efficiency specifically in BRG1-mutated cells, (ii) specifically sensitized BRG1-mutant NSCLC cells toward IR as characterized by a survival reducing factor of 0.63 [95% confidence interval (CI), 0.57-0.69] in the dose range between 2 and 6 Gy, and (iii) decreased the tumor control doses after daily fractionation at 4 Gy in BRG1-mutant NSCLC cell lines A549 and H1299 in minimonolayers by 9.9% ± 1.3% and 13.6% ± 1.8%, respectively. In addition, an increase of residual Rad51-foci at 24 hours after irradiation in BRG1-mutant cells was demonstrated. Therefore, targeting of BRM in combination with radiotherapy is supposed to improve the therapeutic outcome of lung cancer patients harboring BRG1 mutations.The present study shows that the moderate radioresponsiveness of NSCLC cells with BRG1 mutations can be increased upon BRM depletion that is associated with a prolonged Rad51-foci prevalence at DNA DSBs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Terapia Combinada , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/efeitos da radiação , Humanos , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Rad51 Recombinase/genética , Radiação Ionizante , Fatores de Transcrição/antagonistas & inibidores
12.
Clin Cancer Res ; 24(6): 1364-1374, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29298797

RESUMO

Purpose: The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C).Experimental Design: Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms.Results: We identified a 7-gene signature consisting of the three individual genes HILPDA, CD24, TCF3, and one metagene combining the highly correlated genes SERPINE1, INHBA, P4HA2, and ACTN1 The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation (P < 0.001).Conclusions: We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. Clin Cancer Res; 24(6); 1364-74. ©2018 AACR.


Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Transcriptoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiorradioterapia , Criança , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Cuidados Pós-Operatórios , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
13.
Radiother Oncol ; 127(1): 27-35, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29295747

RESUMO

OBJECTIVE: To compare six HPV detection methods in pre-treatment FFPE tumour samples from patients with locally advanced head and neck squamous cell carcinoma (HNSCC) who received postoperative (N = 175) or primary (N = 90) radiochemotherapy. MATERIALS AND METHODS: HPV analyses included detection of (i) HPV16 E6/E7 RNA, (ii) HPV16 DNA (PCR-based arrays, A-PCR), (iii) HPV DNA (GP5+/GP6+ qPCR, (GP-PCR)), (iv) p16 (immunohistochemistry, p16 IHC), (v) combining p16 IHC and the A-PCR result and (vi) combining p16 IHC and the GP-PCR result. Differences between HPV positive and negative subgroups were evaluated for the primary endpoint loco-regional control (LRC) using Cox regression. RESULTS: Correlation between the HPV detection methods was high (chi-squared test, p < 0.001). While p16 IHC analysis resulted in several false positive classifications, A-PCR, GP-PCR and the combination of p16 IHC and A-PCR or GP-PCR led to results comparable to RNA analysis. In both cohorts, Cox regression analyses revealed significantly prolonged LRC for patients with HPV positive tumours irrespective of the detection method. CONCLUSIONS: The most stringent classification was obtained by detection of HPV16 RNA, or combining p16 IHC with A-PCR or GP-PCR. This approach revealed the lowest rate of recurrence in patients with tumours classified as HPV positive and therefore appears most suited for patient stratification in HPV-based clinical studies.


Assuntos
Carcinoma de Células Escamosas/virologia , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/isolamento & purificação , Recidiva Local de Neoplasia/virologia , Infecções por Papillomavirus/virologia , Adulto , Idoso , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Quimiorradioterapia , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Prognóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço
14.
Sci Rep ; 7(1): 13206, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038455

RESUMO

Radiomics applies machine learning algorithms to quantitative imaging data to characterise the tumour phenotype and predict clinical outcome. For the development of radiomics risk models, a variety of different algorithms is available and it is not clear which one gives optimal results. Therefore, we assessed the performance of 11 machine learning algorithms combined with 12 feature selection methods by the concordance index (C-Index), to predict loco-regional tumour control (LRC) and overall survival for patients with head and neck squamous cell carcinoma. The considered algorithms are able to deal with continuous time-to-event survival data. Feature selection and model building were performed on a multicentre cohort (213 patients) and validated using an independent cohort (80 patients). We found several combinations of machine learning algorithms and feature selection methods which achieve similar results, e.g. , MSR-RF: C-Index = 0.71 and BT-COX: C-Index = 0.70 in combination with Spearman feature selection. Using the best performing models, patients were stratified into groups of low and high risk of recurrence. Significant differences in LRC were obtained between both groups on the validation cohort. Based on the presented analysis, we identified a subset of algorithms which should be considered in future radiomics studies to develop stable and clinically relevant predictive models for time-to-event endpoints.

15.
Int J Cancer ; 141(3): 594-603, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28480996

RESUMO

We examined the prognostic role of PD-1+ and CD8+ tumor infiltrating lymphocytes (TILs), and PD-L1+ cells in patients with squamous cell carcinoma of the head and neck (SCCHN) treated with surgery and postoperative chemoradiotherapy (CRT). FFPE samples from 161 patients were immunohistochemically stained for PD-1, CD8 and PD-L1. The immune marker expression was correlated with clinicopathologic characteristics, and overall survival (OS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of HPV16 DNA/p16 status. The median follow-up was 48 months (range: 4-100). The 2-year-OS was 84.1% for the entire cohort. High PD-1 and PD-L1 expression were more common in patients with positive HPV16 DNA (p < 0.001 and p = 0.008, respectively) and high infiltration by CD8+ TILs (p < 0.001 for both markers). High PD-L1 expression correlated with superior OS (p = 0.025), LPFS (p = 0.047) and DMFS (p = 0.048) in multivariable analysis, whereas no significance could be demonstrated for PD-1. Patients with CD8high /PD-L1high expression had favorable outcome (p < 0.001 for all endpoints) compared to other groups. We validated the superior OS data on CD8high /PD-L1high using the Cancer Genome Atlas TCGA dataset (n = 518; p = 0.032). High PD-L1 expression was a favorable prognostic marker in HPV16-negative but not HPV16-positive patients. In conclusion, HPV-positive tumors showed higher expression of immune markers. PD-L1 expression constitutes an independent prognostic marker in SCCHN patients post-adjuvant CRT. In conjunction with CD8 status, these data provide an important insight on the immune contexture of SCCHN and are directly relevant for future treatment stratification with PD-1/PD-L1 immune checkpoint inhibitors to complement CRT.


Assuntos
Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/patologia , Quimiorradioterapia Adjuvante , Neoplasias de Cabeça e Pescoço/patologia , Papillomaviridae/fisiologia , Infecções por Papillomavirus/complicações , Receptor de Morte Celular Programada 1/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Carcinoma de Células Escamosas/virologia , Feminino , Seguimentos , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida
16.
J Cancer Res Clin Oncol ; 143(9): 1733-1744, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28432456

RESUMO

PURPOSE: DNA damage-induced cell death is a major effector mechanism of radiotherapy. Aberrant expression of anti-apoptotic BCL-2 family proteins is frequently observed in lung cancers. Against this background, we studied radioresistance mediated by BCL-2 family proteins at the mechanistic level and its potential as target for radiochemotherapy. METHODS: Lung cancer models stably expressing BCL-xL or MCL-1 were irradiated to study cell death, clonogenic survival, and DNA repair kinetics in vitro, and growth suppression of established tumors in vivo. Additionally, endogenous BCL-xL and MCL-1 were targeted by shRNA or pharmacologic agents prior to irradiation. RESULTS: Radiation exposure induced apoptosis at negligible levels. Yet, anti-apoptotic BCL-xL and MCL-1 expression conferred short-term and long-term radioresistance in vitro and in vivo. Radioresistance correlated with pertubations in homologous recombination repair and repair of DNA double-strand breaks by error-prone, alternative end-joining. Notably, genetic or pharmacologic targeting of BCL-xL or MCL-1 effectively sensitized lung cancer cells to radiotherapy. CONCLUSIONS: In addition to directly suppressing apoptosis, BCL-2 family proteins confer long-term survival benefits to irradiated cancer cells associated with utilization of error-prone repair pathways. Targeting BCL-xL and MCL-1 is an attractive strategy for improving lung cancer radiotherapy.


Assuntos
Reparo do DNA/fisiologia , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tolerância a Radiação/fisiologia , Animais , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
17.
Sci Rep ; 7: 41138, 2017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28112237

RESUMO

Despite good treatment results in localized prostate tumors, advanced disease stages usually have a pronounced resistance to chemotherapy and radiotherapy. The membrane protein caveolin-1 (Cav1) functions here as an important oncogene. Therefore we examined the impact of stromal Cav1 expression for tumor growth and sensitivity to ionizing radiation (IR). Silencing of Cav1 expression in PC3 cells resulted in increased tumor growth and a reduced growth delay after IR when compared to tumors generated by Cav1-expressing PC3 cells. The increased radiation resistance was associated with increasing amounts of reactive tumor stroma and a Cav1 re-expression in the malignant epithelial cells. Mimicking the human situation these results were confirmed using co-implantation of Cav1-silenced PC3 cells with Cav1-silenced or Cav1-expressing fibroblasts. Immunohistochemically analysis of irradiated tumors as well as human prostate tissue specimen confirmed that alterations in stromal-epithelial Cav1 expressions were accompanied by a more reactive Cav1-reduced tumor stroma after radiation and within advanced prostate cancer tissues which potentially mediates the resistance to radiation treatment. Conclusively, the radiation response of human prostate tumors is critically regulated by Cav1 expression in stromal fibroblasts. Loss of stromal Cav1 expression in advanced tumor stages may thus contribute to resistance of these tumors to radiotherapy.


Assuntos
Caveolina 1/metabolismo , Caveolina 1/efeitos da radiação , Progressão da Doença , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Xenoenxertos , Humanos , Masculino , Camundongos , Células Estromais/metabolismo , Células Estromais/efeitos da radiação
18.
Int J Radiat Biol ; 93(5): 494-506, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28044469

RESUMO

PURPOSE: To uncover the role of EZH2 and its opponent ASHL2, a polycomb and trithorax group protein, respectively, on the radioresponsiveness of glioma cell lines. MATERIALS AND METHODS: Expression of EZH2 and ASHL2 was inhibited by siRNA in glioma cell lines. The effect on histone methylation, gene expression, DNA damage repair signaling, cell cycle checkpoints, apoptosis and tumor control were evaluated. RESULTS: Inhibition of EZH2 (EZH2i) led to a transcriptional dysregulation with upregulation of 544 and downregulation of 445 genes. In comparison, ASH2L inhibition (ASH2Li) had an opposed effect with upregulation of 289 and downregulation of 970 genes. EZH2i and ASH2Li significantly reduced methylation of H3K27 and increased methylation of H3K9, respectively. EZH2i and ASH2Li significantly increased and decreased the number of residual γH2AX foci at 24 h after IR, respectively. The former significantly increased radiation-induced cell cycle arrest in G2/M and apoptotic cell death, while ASH2Li decreased both. In addition, a significant shift of the radioresponse curve by -1.22 + 0.23 Gy (p < 0.0001) in the plaque monolayer assay was found after EZH2i in A7 but not in M059K. CONCLUSION: Overall, epigenetic modulation is a promising approach to evaluate the role of chromatin structure for the radioresponsiveness of glioma cell lines.


Assuntos
Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética/genética , Glioma/genética , Glioma/radioterapia , Proteínas Nucleares/genética , Tolerância a Radiação/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Epigênese Genética/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Inativação Gênica/efeitos da radiação , Glioma/patologia , Humanos , Doses de Radiação , Ativação Transcricional/efeitos da radiação
19.
Radiother Oncol ; 121(3): 364-373, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27913065

RESUMO

OBJECTIVE: To investigate the impact of the tumour volume, HPV status, cancer stem cell (CSC) marker expression and hypoxia gene signatures, as potential markers of radiobiological mechanisms of radioresistance, in a contemporary cohort of patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received primary radiochemotherapy (RCTx). MATERIALS AND METHODS: For 158 patients with locally advanced HNSCC of the oral cavity, oropharynx or hypopharynx who were treated at six DKTK partner sites, the impact of tumour volume, HPV DNA, p16 overexpression, p53 expression, CSC marker expression and hypoxia-associated gene signatures on outcome of primary RCTx was retrospectively analyzed. The primary endpoint of this study was loco-regional control (LRC). RESULTS: Univariate Cox regression revealed a significant impact of tumour volume, p16 overexpression, and SLC3A2 and CD44 protein expression on LRC. The tumour hypoxia classification showed a significant impact only for small tumours. In multivariate analyses an independent correlation of tumour volume, SLC3A2 expression, and the 15-gene hypoxia signature with LRC was identified (CD44 protein n/a because of no event in the CD44-negative group). Logistic modelling showed that inclusion of CD44 protein expression and p16 overexpression significantly improved the performance to predict LRC at 2years compared to the model with tumour volume alone. CONCLUSIONS: Tumour volume, HPV status, CSC marker expression and hypoxia gene signatures are potential prognostic biomarkers for patients with locally advanced HNSCC, who were treated by primary RCTx. The study also supports that the individual tumour volumes should generally be included in biomarker studies and that panels of biomarkers are superior to individual parameters.


Assuntos
Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/terapia , Células-Tronco Neoplásicas/metabolismo , Papillomaviridae/isolamento & purificação , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Hipóxia Celular/genética , Quimiorradioterapia , Feminino , Perfilação da Expressão Gênica/métodos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Tolerância a Radiação/genética , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carga Tumoral
20.
Clin Cancer Res ; 22(11): 2639-49, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26755529

RESUMO

PURPOSE: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Expression of the CSC markers CD44, MET, and SLC3A2, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control. RESULTS: Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (P = 0.010) or the 26-gene signature (P = 0.002). In multivariate analyses, in patients with HPV16 DNA-negative but not with HPV16 DNA-positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, P = 0.006; 26-gene signature: HR 10.27, P = 0.024). Furthermore, MET, SLC3A2, CD44, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (MET: HR 3.71, P = 0.016; SLC3A2: HR 8.54, P = 0.037; CD44: HR 3.36, P = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA-negative subgroup. CONCLUSIONS: We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA-negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. Clin Cancer Res; 22(11); 2639-49. ©2016 AACR.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Hipóxia Celular , Quimiorradioterapia , Cisplatino/uso terapêutico , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Papillomavirus Humano 16/genética , Humanos , Receptores de Hialuronatos/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Neoplasias Bucais/terapia , Análise Multivariada , Infecções por Papillomavirus/diagnóstico , Prognóstico , Estudos Prospectivos , Transcriptoma , Resultado do Tratamento
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