RESUMO
A regression-based fusion algorithm has been used to merge hyperspectral Fourier transform infrared (FTIR) data with an H&E image of oral squamous cell carcinoma metastases in cervical lymphoid nodal tissue. This provides insight into the success of the ratio of FTIR absorbances at 1252 cm-1 and 1285 cm-1 in discriminating between these tissue types. The success is due to absorbances at these two wavenumbers being dominated by contributions from DNA and collagen, respectively. A pixel-by-pixel fit of the fused spectra to the FTIR spectra of collagen, DNA and cytokeratin reveals the contributions of these molecules to the tissue at high spatial resolution.
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Carcinoma de Células Escamosas , Neoplasias Bucais , Humanos , Microscopia , Carcinoma de Células Escamosas/patologia , Colágeno , Algoritmos , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
A machine learning algorithm (MLA) has predicted the prognosis of oral potentially malignant lesions and discriminated between lymph node tissue and metastatic oral squamous cell carcinoma (OSCC). The MLA analyses metrics, which are ratios of Fourier transform infrared absorbances, and identifies key wavenumbers that can be associated with molecular biomarkers. The wider efficacy of the MLA is now shown in the more complex primary OSCC tumour setting, where it is able to identify seven types of tissue. Three epithelial and four non-epithelial tissue types were discriminated from each other with sensitivities between 82% and 96% and specificities between 90% and 99%. The wavenumbers involved in the five best discriminating metrics for each tissue type were tightly grouped, indicating that small changes in the spectral profiles of the different tissue types are important. The number of samples used in this study was small, but the information will provide a basis for further, larger investigations.
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Carcinoma de Células Escamosas , Neoplasias Bucais , Humanos , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , AlgoritmosRESUMO
It is shown that a pixel-level image fusion technique can produce images that combine the spatial resolution of optical microscopy images of haematoxylin and eosin (H&E) stained tissue with the chemical information in Fourier transform infrared (FTIR) images. The fused images show minimal distortion and the higher spatial resolution of the H&E images overcomes the diffraction limit on the spatial resolution of the FTIR images. A consideration of the FTIR spectra of nucleic acids and collagen can explain the changes in contrast between non-cancerous oral epithelium and underlying stroma within fused images formed by combining an H&E stain of oral tissue with FTIR images of the tissue obtained at a number of wavenumbers.
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Tecido Conjuntivo , Microscopia , Colágeno , Análise de Fourier , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
A novel machine learning algorithm is shown to accurately discriminate between oral squamous cell carcinoma (OSCC) nodal metastases and surrounding lymphoid tissue on the basis of a single metric, the ratio of Fourier transform infrared (FTIR) absorption intensities at 1252 cm-1 and 1285 cm-1. The metric yields discriminating sensitivities, specificities and precision of 98.8 ± 0.1%, 99.89 ± 0.01% and 99.78 ± 0.02% respectively, and an area under receiver operator characteristic (AUC) of 0.9935 ± 0.0006. The delineation of the OSCC and lymphoid tissue revealed by the image formed from the metric is in better agreement with an immunohistochemistry (IHC) stained image than are either of the FTIR images obtained at the individual wavenumbers. Scanning near-field optical microscopy (SNOM) images of the tissue obtained at a number of key wavenumbers, with high spatial resolution, show variations in the chemical structure of the tissue with a feature size down to â¼4 µm. The image formed from the ratio of the SNOM images obtained at 1252 cm-1 and 1285 cm-1 shows more contrast than the SNOM images obtained at these or a number of other individual wavenumbers. The discrimination between the two tissue types is dominated by the contribution from the 1252 cm-1 signal, which is representative of nucleic acids, and this shows the OSCC tissue to be accompanied by two wide arcs of tissue which are particularly low in nucleic acids. Haematoxylin and eosin (H&E) staining shows the tumour core in this specimen to be â¼40 µm wide and the SNOM topography shows that the core centre is raised by â¼1 µm compared to the surrounding tissue. Line profiles of the SNOM signal intensity taken through the highly keratinised core show that the increase in height correlates with an increase in the protein signal. SNOM line profiles show that the nucleic acids signal decreases at the centre of the tumour core between two peaks of higher intensity. All these nucleic acid features are â¼25 µm wide, roughly the width of two cancer cells.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Algoritmos , Humanos , Microscopia , Neoplasias Bucais/diagnóstico , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
iRhom2 is an inactive rhomboid protease involved in diverse signalling events. It has been implicated in the pathogenesis of a number of cancer types, including oesophageal and ovarian cancer, while its closely associated family member, iRhom1, is implicated in head and neck cancer. However, a role for iRhom2 in head and neck cancer has not been investigated. Immunoblotting for iRhom2 in 54 oral squamous cell carcinoma (OSCC) and 24 paired normal tissues demonstrated higher levels of iRhom2 protein in tumour compared with normal samples (P < 0.05). iRhom2 over-expression correlated with poor patient survival (P < 0.0005) but with no other clinicopathological variable. Increased cell migration was observed in stably over-expressing iRhom2 clones of OSCC cell lines in the absence of increased cell proliferation, but not in the normal oral keratinocyte cell line, NOK-hTERT, and this was abrogated by knock-down of iRhom2. iRhom2 protein expression is increased in a proportion of OSCC and this up-regulation is associated with faster cell migration and decreased patient survival. These data implicate iRhom2-controlled signalling events in the pathogenesis of this cancer.
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Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Bucais/patologia , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
BACKGROUND: Taxanes are mitotic poisons widely used in the treatment of non-small cell lung cancer (NSCLC), however, little is known about potential molecular modulators of response to these compounds. Aurora B (AURKB) is a critical regulator of the mitotic spindle assembly, previously shown overexpressed in NSCLC. Here we investigated the hypothesis that AURKB expression modulates the efficacy of taxanes in NSCLC cells. METHODS: AURKB mRNA expression was determined by qPCR in 132 frozen NSCLC tissues and nine NSCLC cell lines. Aurora B expression was knocked down in cell lines using multiple shRNA constructs. Barasertib was used to specifically inhibit AURKB activity, determined by the level of H3S10 phosphorylation. RESULTS: Frequent AURKB mRNA upregulation was observed in NSCLC tissues (P<0.0001), being more prominent in squamous carcinomas (P<0.0001). Aurora B expression in cell lines strongly correlated with sensitivity to both docetaxel (P=0.004) and paclitaxel (P=0.007). Aurora B knockdown derivatives consistently showed a dose-dependent association between low-AURKB expression and resistance to paclitaxel. Specific chemical inhibition of Aurora B activity also demonstrated a strong dose-dependent efficiency in triggering paclitaxel resistance. CONCLUSIONS: Aurora B activity is an important modulator of taxane response in NSCLC cells. This may lead to further insights into taxane sensitivity of NSCLC tumours.
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Antineoplásicos Fitogênicos/uso terapêutico , Aurora Quinase B/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Organofosfatos/farmacologia , Quinazolinas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Tylosis esophageal cancer (TOC) is an autosomal-dominant syndrome characterized by palmoplantar keratoderma, oral precursor lesions, and a high lifetime risk of esophageal cancer. We have previously localized the TOC locus to a small genomic interval within chromosomal region 17q25. Using a targeted capture array and next-generation sequencing, we have now identified missense mutations (c.557T>C [p.Ile186Thr] and c.566C>T [p.Pro189Leu] in RHBDF2, which encodes the inactive rhomboid protease RHBDF2 (also known as iRhom2), as the underlying cause of TOC. We show that the distribution of RHBDF2 in tylotic skin is altered in comparison with that in normal skin, and immortalized tylotic keratinocytes have decreased levels of total epidermal growth factor receptor (EGFR) and display an increased proliferative and migratory potential relative to normal cells, even when normal cells are stimulated with exogenous epidermal growth factor. It would thus appear that EGFR signaling is dysregulated in tylotic cells. Furthermore, we also show an altered localization of RHBDF2 in both tylotic and sporadic squamous esophageal tumors. The elucidation of a role of RHBDF2 in growth-factor signaling in esophageal cancer will help to determine whether targeting this pathway in chemotherapy for this and other squamous cell carcinomas will be effective.
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Neoplasias Esofágicas/genética , Ceratodermia Palmar e Plantar Difusa/genética , Mutação de Sentido Incorreto , Serina Proteases/genética , Sequência de Aminoácidos , Carcinoma de Células Escamosas/genética , Processos de Crescimento Celular/genética , Movimento Celular/genética , Cromossomos Humanos Par 17/genética , Receptores ErbB/genética , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Éxons , Humanos , Queratinócitos/metabolismo , Ceratodermia Palmar e Plantar Difusa/enzimologia , Ceratodermia Palmar e Plantar Difusa/metabolismo , Ceratodermia Palmar e Plantar Difusa/patologia , Dados de Sequência Molecular , Linhagem , Fenótipo , Alinhamento de Sequência , Serina Endopeptidases , Regiões não TraduzidasRESUMO
In this study, optical photothermal infrared (O-PTIR) spectroscopy combined with machine learning algorithms were used to evaluate 46 tissue cores of surgically resected cervical lymph nodes, some of which harboured oral squamous cell carcinoma nodal metastasis. The ratios obtained between O-PTIR chemical images at 1252 cm-1 and 1285 cm-1 were able to reveal morphological details from tissue samples that are comparable to the information achieved by a pathologist's interpretation of optical microscopy of haematoxylin and eosin (H&E) stained samples. Additionally, when used as input data for a hybrid convolutional neural network (CNN) and random forest (RF) analyses, these yielded sensitivities, specificities and precision of 98.6 ± 0.3%, 92 ± 4% and 94 ± 5%, respectively, and an area under receiver operator characteristic (AUC) of 94 ± 2%. Our findings show the potential of O-PTIR technology as a tool to study cancer on tissue samples.
Assuntos
Carcinoma de Células Escamosas , Metástase Linfática , Neoplasias Bucais , Humanos , Metástase Linfática/patologia , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/patologia , Linfonodos/patologia , Espectrofotometria Infravermelho/métodos , Aprendizado de Máquina , Redes Neurais de Computação , Feminino , Masculino , Curva ROCRESUMO
BACKGROUND: Estimation of prognosis of oral squamous cell carcinoma (OSCC) is inaccurate prior to surgery, only being effected following subsequent pathological analysis of the primary tumour and excised lymph nodes. Consequently, a proportion of patients are overtreated, with an increase in morbidity, or undertreated, with inadequate margins and risk of recurrence. We hypothesise that it is possible to accurately characterise clinical outcomes from infrared spectra arising from diagnostic biopsies. In this first step, we correlate survival with IR spectra derived from the primary tumour. METHODS: Infrared spectra were collected from tumour tissue from 29 patients with OSCC and subject to classification modelling. RESULTS: The model had a median AUROC of 0.89 with regard to prognosis, a median specificity of 0.83, and a hazard ratio of 6.29 in univariate Cox proportional hazard modelling. CONCLUSION: The data suggest that FTIR spectra may be a useful early biomarker of prognosis in OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , PrognósticoRESUMO
Background and Aims: Survival rates for esophageal squamous cell carcinoma (ESCC) are extremely low due to the late diagnosis of most cases. An understanding of the early molecular processes that lead to ESCC may facilitate opportunities for early diagnosis; however, these remain poorly defined. Tylosis with esophageal cancer (TOC) is a rare syndrome associated with a high lifetime risk of ESCC and germline mutations in RHBDF2, encoding iRhom2. Using TOC as a model of ESCC predisposition, this study aimed to identify early-stage transcriptional changes in ESCC development. Methods: Esophageal biopsies were obtained from control and TOC individuals, the latter undergoing surveillance endoscopy, and adjacent diagnostic biopsies were graded as having no dysplasia or malignancy. Bulk RNA-Seq was performed, and findings were compared with sporadic ESCC vs normal RNA-Seq datasets. Results: Multiple transcriptional changes were identified in TOC samples, relative to controls, and many were detected in ESCC. Accordingly, pathway analyses predicted an enrichment of cancer-associated processes linked to cellular proliferation and metastasis, and several transcription factors were predicted to be associated with TOC and ESCC, including negative enrichment of GRHL2. Subsequently, a filtering strategy revealed 22 genes that were significantly dysregulated in both TOC and ESCC. Moreover, Keratin 17, which was upregulated in TOC and ESCC, was also found to be overexpressed at the protein level in 'normal' TOC esophagus tissue. Conclusion: Transcriptional changes occur in TOC esophagus prior to the onset of dysplasia, many of which are associated with ESCC. These findings support the utility of TOC to help reveal the early molecular processes that lead to sporadic ESCC.
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BACKGROUND: Local recurrence in oral squamous cell carcinoma (OSCC) despite clear surgical margins may indicate the presence of residual, sub-microscopic disease. Molecular assessment of surgical margins may provide a greater prognostic sensitivity compared to histopathology. We aimed to determine whether promoter methylation in deep and mucosal resection margins can predict recurrence in OSCC. METHODS: Forty-eight consecutive OSCC cases were recruited and a 5 mm(3) tumor sample plus 5 deep and 5 mucosal margin samples were snap frozen. Clinical, pathological, adjuvant therapy, and outcome data were recorded. Tumors were informative if >5 % promoter methylation was found for ≥1 of 4 genes using qMSP. Margins were declared molecularly positive if >1 % promoter methylation was found in any margin. RESULTS: Thirty (63 %) of 48 cases were methylation informative. Mucosal margin samples were largely positive for methylation (26 of 30, 87 %), indicating the presence of field cancerization. Methylation at ≥1 gene promoters in ≥1 deep margin correlated with the presence of close/involved mucosal margins (P = 0.027) and increased pT status (P = 0.027) but not the status of deep margins, recurrence, or survival. CONCLUSIONS: The current gene panel did not add prognostic information to histopathological reporting of resection margins. Future efforts should concentrate on improving gene selection, informativity, and assay performance in the patient group with intermediate indications for adjuvant therapy.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Bucais/genética , Recidiva Local de Neoplasia/genética , Regiões Promotoras Genéticas , Idoso , Caderinas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Citoglobina , Feminino , Genes p16 , Globinas/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Neoplasias Bucais/cirurgia , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/patologia , Neoplasia ResidualRESUMO
OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. STUDY DESIGN: Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. RESULTS: In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. CONCLUSIONS: Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.
Assuntos
Carcinoma de Células Escamosas , Carcinoma Verrucoso , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Metilação de DNA/genética , Leucoplasia Oral/genética , Leucoplasia Oral/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Mutação/genética , Transformação Celular Neoplásica/patologia , Carcinoma Verrucoso/patologiaRESUMO
OBJECTIVE: Predicting malignant transformation (MT) in oral epithelial dysplasia (OED) is challenging. The higher rate of MT reported in nonsmokers suggests an endogenous etiology in oncogenesis. We hypothesize that loss of FANCD2 and associated proteins could influence genomic instability and MT in the absence of environmental carcinogens. STUDY DESIGN: Longitudinal archival samples were obtained from 40 individuals with OED: from diagnosis to the most recent review in 23 patients with stable OED or until excision of the squamous cell carcinoma in 17 patients with unstable OED undergoing MT. Histopathological reassessment, immunohistochemistry for FANCD2, and Western blotting for phosphorylation/monoubiquitylation status of ATR, CHK1, FANCD2, and FANCG were undertaken on each tissue sample. RESULTS: Decreased expression of FANCD2 was observed in the diagnostic biopsies of OED lesions that later underwent MT. Combining the FANCD2 expression scores with histologic grading more accurately predicted MT (P = .005) than histology alone, and it correctly predicted MT in 10 of 17 initial biopsies. Significantly reduced expression of total FANCD2, pFANCD2, pATR, pCHK-1, and pFANCG was observed in unstable OED. CONCLUSIONS: There is preliminary evidence that defects in the DNA damage sensing/signaling/repair cascade are associated with MT in OED. Loss of expression of FANCD2 protein in association with a higher histologic grade of dysplasia offered better prediction of MT than clinicopathologic parameters alone.
Assuntos
Carcinoma de Células Escamosas , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Neoplasias Bucais , Lesões Pré-Cancerosas , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Humanos , Hiperplasia , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
A machine learning algorithm (MLA) has been applied to a Fourier transform infrared spectroscopy (FTIR) dataset previously analysed with a principal component analysis (PCA) linear discriminant analysis (LDA) model. This comparison has confirmed the robustness of FTIR as a prognostic tool for oral epithelial dysplasia (OED). The MLA is able to predict malignancy with a sensitivity of 84 ± 3% and a specificity of 79 ± 3%. It provides key wavenumbers that will be important for the development of devices that can be used for improved prognosis of OED.
RESUMO
Oral epithelial dysplasia (OED) is a histopathologically-defined, potentially premalignant condition of the oral cavity. The rate of transformation to frank carcinoma is relatively low (12% within 2 years) and prediction based on histopathological grade is unreliable, leading to both over- and under-treatment. Alternative approaches include infrared (IR) spectroscopy, which is able to classify cancerous and non-cancerous tissue in a number of cancers, including oral. The aim of this study was to explore the capability of FTIR (Fourier-transform IR) microscopy and machine learning as a means of predicting malignant transformation of OED. Supervised, retrospective analysis of longitudinally-collected OED biopsy samples from 17 patients with high risk OED lesions: 10 lesions transformed and 7 did not over a follow-up period of more than 3 years. FTIR spectra were collected from routine, unstained histopathological sections and machine learning used to predict malignant transformation, irrespective of OED classification. PCA-LDA (principal component analysis followed by linear discriminant analysis) provided evidence that the subsequent transforming status of these 17 lesions could be predicted from FTIR data with a sensitivity of 79 ± 5% and a specificity of 76 ± 5%. Six key wavenumbers were identified as most important in this classification. Although this pilot study used a small cohort, the strict inclusion criteria and classification based on known outcome, rather than OED grade, make this a novel study in the field of FTIR in oral cancer and support the clinical potential of this technology in the surveillance of OED.
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Neoplasias Bucais , Lesões Pré-Cancerosas , Transformação Celular Neoplásica/patologia , Humanos , Hiperplasia , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/patologia , Projetos Piloto , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/patologia , Estudos Retrospectivos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
BACKGROUND/AIM: Utilising radiotherapy in the management of head and neck cancer (HNC) often results in long term toxicities. Mandibular osteoradionecrosis (ORN) represents a late toxicity associated with significant morbidity. We aim to identify a panel of common genetic variants which can predict ORN to aid development of personalised radiotherapy protocols. METHOD: Single nucleotide polymorphism (SNP) arrays were applied to DNA samples from patients who had prior HNC radiotherapy and minimum two years follow-up. A case cohort of mandibular ORN was compared to a control group of participants recruited to CRUK HOPON clinical trial. Relevant clinical parameters influencing ORN risk (e.g. smoking/alcohol) were collected. Significant associations from array data were internally validated using polymerase chain reaction (PCR) and pyrosequencing. RESULTS: Following inclusion of 141 patients in the analysis (52 cases, 89 controls), a model predictive for ORN was developed; after controlling for alcohol consumption, smoking, and age, 4053 SNPs were identified as significant. This was reduced to a representative model of 18 SNPs achieving 92% accuracy. Following internal technical validation, a six SNP model (rs34798038, rs6011731, rs2348569, rs530752, rs7477958, rs1415848) was retained within multivariate regression analysis (ROC AUC 0.859). Of these, four SNPs (rs34798038 (A/G) (p 0.006), rs6011731 (C/T) (p 0.018), rs530752 (A/G) (p 0.046) and rs2348569 (G/G) (p 0.005)) were significantly associated with the absence of ORN. CONCLUSION: This is the first genome wide association study in HNC using ORN as the endpoint and offers new insight into ORN pathogenesis. Subject to validation, these variants may guide patient selection for personalised radiotherapy strategies.
Assuntos
Neoplasias de Cabeça e Pescoço , Osteorradionecrose , Estudos de Coortes , Estudo de Associação Genômica Ampla , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Mandíbula , Osteorradionecrose/genética , Estudos RetrospectivosRESUMO
HURP gene encodes the hepatoma upregulated protein (HURP), a microtubule associated protein regulating mitotic spindle dynamics, which promotes chromosomal congression and alignment during mitosis, with a potential role in tumorigenesis. In the present study, HURP mRNA expression was investigated by reverse transcription-quantitative PCR in oropharyngeal squamous cell carcinoma (OPSCC). Primary OPSCC tumors from 107 patients and 48 adjacent normal tissues, as well as 12 respiratory tract cancer cell lines (9 head and neck squamous cell carcinoma, 2 lung cancer and 1 normal bronchial) were utilised in the present study. mRNA expression levels of HURP were higher in malignant OPSCC tissues compared with in normal mucosa (P<1×10-5) and significantly associated with sex and smoking status (P<0.0001). Vinorelbine in vitro toxicity at half-maximal inhibitory concentration (IC50) was measured in the 11 cancer cell lines using an MTT assay. Sensitivity to vinorelbine was significantly correlated with HURP expression (r=0.636; P=0.035). The data indicated that HURP overexpression is frequent in OPSCC tissues and associated with smoking. The correlation between HURP mRNA expression and vinorelbine in vitro response suggests that HURP is a potential modulator of vinorelbine response; therefore, it should be explored for its possible predictive value for the efficiency of vinorelbine treatment in this type of cancer.
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OBJECTIVES: The incidence of human papillomavirus (HPV)-positive head and neck cancer, particularly oropharyngeal, has been increasing rapidly. Understanding of this disease, and modelling of suitable therapeutics, requires sustainable cell cultures, yet they remain limited in number and of variable origin. A comprehensive understanding of these resources is therefore of great importance. MATERIALS AND METHODS: Viral gene expression assays and pathological testing methods were used in the six currently available HPV-positive cell lines derived from head and neck (H&N) subsites, two HPV-negative H&N and two cervical carcinoma cell lines. A 2D migration assay monitored cell movement, speed and pattern of migration. RESULTS: All six H&N and two cervical cell lines were confirmed HPV-positive by gold standard testing, yet variability between tests was apparent. Although migration was not significantly different between cell lines, each demonstrated unique migration patterns. CONCLUSION: Patient-derived cancer cells, arising as a consequence of natural oncogenic processes rather than in vitro manipulation, are essential for understanding cancer biology. We have characterised the available HPV-positive H&N cell lines and provided clear evidence of a persisting viral oncogenic driver in each, as such supporting their ongoing use as a model of HPV-positive H&N cancer. Importantly, we also highlight a need for caution to be exercised when translating future in vitro findings associated with these lines particularly in the context of oropharyngeal cancer given irregularities in tumour provenance (origin site and clinicopathological features).
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Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/virologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/complicações , Linhagem Celular Tumoral , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Infecções por Papillomavirus/virologiaRESUMO
A de-waxing protocol that successfully removes paraffin from tissue microarray (TMA) cores of fixed tissue obtained from oral cancer is described. The success of the protocol is demonstrated by the comparison of Fourier transform infrared (FTIR) results obtained on paraffin-embedded and de-waxed tissue and the absence of any significant correlations between infrared scanning near-field optical microscopy (SNOM) images of de-waxed tissue obtained at the three main paraffin IR peaks. The success of the protocol in removing paraffin from tissue is also demonstrated by images obtained with scanning electron microscopy (SEM) and by energy dispersive spectra (EDS) of a de-waxed CaF2 disc which shows no significant contribution from carbon. The FTIR spectra of the de-waxed TMA core overlaps that obtained from OE19 oesophageal cancer cells which had never been exposed to paraffin.
Assuntos
Microscopia de Varredura por Sonda , Parafina , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier , CerasRESUMO
We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.