RESUMO
Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.
Assuntos
Carcinoma de Células Escamosas , Papillomavirus Humano , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Integração Viral , Feminino , Humanos , Masculino , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Linhagem Celular Tumoral , DNA Viral/genética , Formaldeído , Papillomavirus Humano/classificação , Papillomavirus Humano/genética , Papillomavirus Humano/isolamento & purificação , Neoplasias Orofaríngeas/virologia , Neoplasias Orofaríngeas/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/diagnóstico , Inclusão em Parafina , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Fixação de Tecidos , Integração Viral/genéticaRESUMO
Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemistry and results were correlated with CD8+ T-cell inflammation and human papillomavirus (HPV)-status. 73 OPSCC stained positive for LAG-3 (31%; HPV+:44%; HPV-:26%, p = 0.006), 122 OPSCC stained positive for TIM-3 (51%; HPV+:70%; HPV-:44%, p < 0.001) and 168 OPSCC (70%; HPV+:75%; HPV-:68%, p = 0.313) for VISTA. CD8+ T-cells were significantly associated with LAG-3, TIM-3 and VISTA expression (p < 0.001, p < 0.001, p = 0.007). Immune checkpoint therapy targeting LAG-3, TIM-3, and/or VISTA could be a promising treatment strategy especially in HPV-related OPSCC. Future clinical trials investigating the efficacy of a checkpoint blockade in consideration of LAG-3, TIM-3, and VISTA expression are required.
Assuntos
Antígenos CD/metabolismo , Antígenos B7/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Orofaríngeas/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Estudos de Casos e Controles , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Orofaríngeas/imunologia , Neoplasias Orofaríngeas/metabolismo , Prognóstico , Taxa de Sobrevida , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (OPSCC) differs from HPV-negative (-) OPSCC. HPV-related immune-escape-mechanism could be responsible for the development and progression of HPV+ tumors and an immunophenotype different from HPV- OPSCC is expected. The purpose of this study was to analyze the expression of programmed cell death protein 1 ligand 1 (PD-L1) and its prognostic relevance in relation to CD8+ tumor infiltrating lymphocytes (TILs) and the major histocompatibility complex (MHC) I expression in OPSCC. We quantified PD-L1 expression on tumor cells (TC) and macrophages and MHC I expression in association to CD8+ TILs by immunohistochemistry on tissue microarray derived from 171 HPV+/-OPSCC. HPV-status was determined by p16INK4a immunohistochemistry/HPV-DNA detection. Presence of CD8+ TILs, PD-L1 expression on TC, and a more frequent loss of MHC I in HPV+ compared to HPV- OPSCC was detected. A high amount of CD8+ TILs in the whole cohort and in HPV+ OPSCC and PD-L1 expression on TC in HPV- OPSCC was associated with favorable overall survival. There was a trend for an improved outcome according to PD-L1 expression (macrophages) in HPV+ OPSCC without reaching statistical significance. CD8+ TILs and PD-L1-expression have prognostic impact in OPSCC and might present useful biomarkers for predicting clinical outcome and personalized therapy concepts.
Assuntos
Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Linfócitos T CD8-Positivos , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias de Cabeça e Pescoço , Linfócitos do Interstício Tumoral , Proteínas de Neoplasias/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Intervalo Livre de Doença , Feminino , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Taxa de SobrevidaRESUMO
Different studies have shown that HPV16-positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome-wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty-three fresh-frozen HPV-16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo-keto-reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV-positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non-hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV-positive (p ≤0.001) and -negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV-negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.
Assuntos
20-Hidroxiesteroide Desidrogenases/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/genética , Neoplasias Orofaríngeas/genética , Regulação para Cima/genética , Integração Viral/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , DNA Viral/genética , Feminino , Genes Virais/genética , Humanos , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Prognóstico , Taxa de SobrevidaRESUMO
Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/secundário , MicroRNAs/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Orofaríngeas/patologia , Infecções por Papillomavirus/complicações , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Células Cultivadas , Feminino , Seguimentos , Perfilação da Expressão Gênica , Papillomavirus Humano 16/isolamento & purificação , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/virologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/virologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/virologia , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , PrognósticoRESUMO
HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Carga Viral , Integração Viral/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , DNA Viral/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
BACKGROUND: The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/virologia , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genéticaRESUMO
It has been shown that podoplanin expression is associated with carcinoma of the aerodigestive tract. Recent studies indicate that podoplanin may serve as a prognostic biomarker in oral carcinoma. In order to provide evidence on the role of podoplanin in oropharyngeal squamous cell carcinoma, we evaluated the prognostic impact of podoplanin in these patients. We analyzed formalin-fixed tissue samples from 107 consecutive patients with oropharyngeal squamous cell carcinoma. HPV typing and immunohistochemical staining for both p16 and podoplanin were performed. Expression of podoplanin was seen in 38.3% of all cases. We found no correlation of the podoplanin scores with either p16 expression or with HPV status. There was no significant correlation of podoplanin expression with the staging variables T, N, M, and tumor grading. Podoplanin expression did neither influence the 5-year overall survival nor the 5-year disease-free survival. Concluding, we could not find a prognostic role of podoplanin expression neither in the HPV-positive cases nor in the HPV-negative cases. It appears that podoplanin is not expressed as often in oropharyngeal cancer compared to oral cancer. We could not show any relation of lymph node metastases and podoplanin expression in this homogenous cohort of tumors.
Assuntos
Carcinoma de Células Escamosas , Glicoproteínas de Membrana/metabolismo , Neoplasias Bucais , Neoplasias Orofaríngeas , Adulto , Idoso , Biomarcadores , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Papillomaviridae/isolamento & purificação , PrognósticoRESUMO
Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence.
Assuntos
Carcinoma de Células Escamosas/virologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Neoplasias Orofaríngeas/virologia , Papiloma/virologia , Infecções por Papillomavirus/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/metabolismo , Criança , Pré-Escolar , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lactente , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/virologia , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/metabolismo , Papiloma/metabolismo , Infecções por Papillomavirus/complicações , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/virologia , Fatores de Risco , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto JovemRESUMO
Tonsillar squamous cell carcinoma (TSCC) is frequently associated with human papillomavirus (HPV) and chromosome instability. Data from cellular model systems are, however, controversial concerning a relation between HPV and chromosome instability development. Here we studied this association in 77 primary TSCC with known clinical outcome and cell cycle protein expression profiles. Thirty-two tumors (42%) showed HPV16-integration. All 77 cases were analyzed by fluorescence in situ hybridization using chromosome 1- and 7-specific centromere DNA probes to detect chromosome instability, indicated by the presence of chromosome imbalances and/or polyploidization for these chromosomes. In addition, eight HPV-positive dysplasias, seven of which were adjacent to a carcinoma, were analyzed. Disomy for chromosome 1 and 7 was present in 29 out of 77 TSCC (38%), of which 19 were HPV16-positive (p = 0.002). Aneusomy was observed in the remaining 48 TSCC, of which 13 were HPV-positive. Aneusomies correlated significantly with tobacco- and alcohol consumption (p = 0.001 and p = 0.016, respectively) and a higher T-stage (p = 0.018). Both HPV-positivity and chromosome disomy were significantly associated with a favorable disease-free survival (p = 0.001 and p = 0.025, respectively). Particularly in the HPV16-positive group chromosome instability is a very strong indicator for an unfavorable prognosis (p = 0.032). In the dysplasias an identical HPV and chromosome copy number status was identified as in the adjacent tumors. We conclude that HPV-positive TSCC and their precursor lesions are more often genetically stable than HPV-negative lesions and that these tumors are associated with a favorable prognosis. Chromosome instability is an indicator for unfavorable prognosis, particularly in the HPV-positive patient group.
Assuntos
Carcinoma de Células Escamosas/genética , Instabilidade Cromossômica , Papillomavirus Humano 16/genética , Neoplasias Tonsilares/genética , Integração Viral , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Sondas de DNA , Humanos , Hibridização in Situ Fluorescente , Prognóstico , Neoplasias Tonsilares/patologia , Neoplasias Tonsilares/virologiaRESUMO
Although cell proliferation is related to tumour aggressiveness and prognosis, there are few studies describing the expression of proliferative markers in salivary gland cancer. Our aim was to assess the long-term prognostic value of the proliferating cell nuclear antigen (PCNA) in a large group of histologically different salivary gland cancers. We analysed the expression of PCNA in 159 patients with parotid gland cancer by means of immunohistochemistry. The mean follow-up time was 56.6 months. A high expression of PCNA showed a significant correlation to the patients' pathological lymph node stage (p = 0.004). A high PCNA expression significantly indicated a poor 5-year disease-free (p = 0.046) and overall survival rate (p = 0.018). The PCNA expression was the only prognostic factor for a worse 5-year disease-free and overall survival in acinic cell carcinomas (p = 0.004, p = 0.022). The correlation between PCNA expression and survival probabilities of salivary gland cancer might make proliferation markers helpful tools in patient follow-up, prognosis and targeted therapy in salivary gland cancer in future.
Assuntos
Carcinoma de Células Acinares/metabolismo , Glândula Parótida/química , Neoplasias Parotídeas/metabolismo , Antígeno Nuclear de Célula em Proliferação/biossíntese , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Acinares/patologia , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Glândula Parótida/patologia , Neoplasias Parotídeas/diagnóstico , Neoplasias Parotídeas/mortalidade , Prognóstico , Taxa de SobrevidaRESUMO
AIMS: High-risk human papillomaviruses (HPVs) constitute an important risk factor for tonsillar cancer. This study describes changes in cell adhesion molecules during metastasis of HPV-related and HPV-unrelated tonsillar carcinomas. METHODS AND RESULTS: We examined 48 primary tonsillar carcinoma samples (25 HPV-16 DNA-positive, 23 HPV-16 DNA-negative) and their respective lymph node metastases for their HPV status and for the expression of p16, epithelial cadherin (E-cadherin), ß-catenin, and vimentin. A positive HPV-specific polymerase chain reaction finding correlated significantly with p16 overexpression in both primary tumours and their metastases (P<0.0001 for both). In HPV-unrelated carcinomas, the expression of E-cadherin was significantly lower in metastases than in primary tumours (P<0.001). In contrast, the expression of nuclear ß-catenin was significantly higher in metastases than in primary tumours (P=0.016). In HPV-related carcinomas, nuclear localization of ß-catenin expression was already apparent in primary tumours (P=0.030). The expression of vimentin significantly correlated with the grading of the primary tumour (P=0.021). CONCLUSIONS: Our data indicate that the down-regulation of E-cadherin and the up-regulation of nuclear ß-catenin expression might be crucial steps during tumour progression of tonsillar carcinomas, being already present in primary tumours in HPV-driven carcinomas, but becoming apparent in HPV-unrelated tumours later in the process of metastasis.
Assuntos
Caderinas/metabolismo , Carcinoma de Células Escamosas/secundário , Núcleo Celular/metabolismo , Infecções por Papillomavirus/patologia , Neoplasias Tonsilares/patologia , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Linfonodos/virologia , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/metabolismo , Neoplasias Tonsilares/metabolismo , Neoplasias Tonsilares/virologiaRESUMO
VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.
RESUMO
PURPOSE: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. EXPERIMENTAL DESIGN: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16(INK4A) immunostaining. The results were correlated with HPV status and clinical data from patients. RESULTS: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P=0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P=0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P=0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P=0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P=0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P=0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P=0.008; disease-free survival, P=0.01) and none of these patients had a tumor recurrence. CONCLUSIONS: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Perfilação da Expressão Gênica , Papillomavirus Humano 16/genética , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/metabolismo , Consumo de Bebidas Alcoólicas , Carcinoma de Células Escamosas/virologia , Aberrações Cromossômicas , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 3/genética , Hibridização Genômica Comparativa , Estudos de Viabilidade , Dosagem de Genes , Papillomavirus Humano 16/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Estadiamento de Neoplasias , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Prognóstico , Fatores de Risco , Fumar , Taxa de SobrevidaRESUMO
Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and CD36 as targets of viral integration are involved in cell migration and ECM-receptor interactions, which provides a possible mechanism for clonal migration from one tonsil to another.
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B lymphocytes are important players in immune responses to cancer. However, their composition and function in head and neck squamous cell carcinoma (HNSCC) has not been well described. Here, we analyzed B cell subsets in HNSCC (n = 38), non-cancerous mucosa (n = 14) and peripheral blood from HNSCC patients (n = 38) and healthy controls (n = 20) by flow cytometry. Intratumoral B cells contained high percentages of activated (CD86+), antigen-presenting (CD86+/CD21-) and memory B cells (IgD-/CD27+). T follicular helper cells (CD4+/CXCR5+/CD45RA-/CCR7-) as key components of tertiary lymphoid structures and plasma cells made up high percentages of the lymphocyte infiltrate. Percentages of regulatory B cell varied depending on the regulatory phenotype. Analysis of humoral immune responses against 23 tumor-associated antigens (TAA) showed reactivity against at least one antigen in 56% of HNSCC patients. Reactivity was less frequent in human papillomavirus associated (HPV+) patients and healthy controls compared to HPV negative (HPV-) HNSCC. Likewise, patients with early stage HNSCC or MHC-I loss on tumor cells had low TAA responses. Patients with TAA responses showed CD4+ dominated T cell infiltration compared to mainly CD8+ T cells in tumors without detected TAA response. To summarize, our data demonstrates different immune infiltration patterns in relation to serological TAA response detection and the presence of B cell subpopulations in HNSCC that can engage in tumor promoting and antitumor activity. In view of increasing use of immunotherapeutic approaches, it will be important to include B cells into comprehensive phenotypic and functional analyses of tumor-associated lymphocytes.
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A hallmark of solid tumors is the consumption of large amounts of glucose and production of lactate, also known as Warburg-like metabolism. This metabolic phenotype is typical for aggressive tumor growth, and can be visualized by 18F-fluorodeoxyglucose (18F-FDG) uptake detected by positron emission tomography (PET). High 18F-FDG uptake inversely correlates with survival and goes along with reduced expression of the catalytic beta-subunit of the H+-ATP synthase (ß-F1-ATPase) in several tumor entities analyzed so far.For this study we characterized a series of 15 head and neck squamous cell carcinoma (HNSCC) by (i) determining 18F-FDG-uptake; (ii) quantitative expression analysis of ß-F1-ATPase (Complex V), NDUF-S1 (Complex I) and COX1 (Complex IV) of the mitochondrial electron transport chain (ETC), as well as Hsp60 (mitochondrial mass) and GAPDH (glycolysis) in tumor cells; (iii) sequencing of the mtDNA of representative tumor samples.Whereas high 18F-FDG-uptake also correlates with poor prognosis in HNSCC, it surprisingly is accompanied by high levels of ß-F1-ATPase, but not by any of the other analyzed proteins.In conclusion, we here describe a completely new phenotype of metabolic adaptation possibly enabling those tumors with highest levels of ß-F1-ATPase to rapidly proliferate even in hypoxic zones, which are typical for HNSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Glucose/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/enzimologia , Feminino , Fluordesoxiglucose F18/análise , Fluordesoxiglucose F18/farmacocinética , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/enzimologia , Tomografia por Emissão de Pósitrons/métodos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
BACKGROUND: Oncogenic human papillomaviruses (HPV) are known to be associated with carcinomas of the uterine cervix. Furthermore, current studies have shown that HPV-infection is also associated with a subtype of oropharyngeal cancers. In general, a sexual transmission of the viruses has been shown by numerous studies in the genital lesions. However, there are unknown factors regarding the prevalence and transmission of HPV in the oropharynx. The aim of this study was to evaluate HPV prevalence in the oropharynx in female participants with and without genital HPV infection. In addition, we analyzed risk factors for an oropharyngeal colonization with HPV in their sexual partners, too. METHODS: 129 Female participants were tested for presence of HPV-DNA by oral lavage, brush cytology of the tonsils and of the cervix. In addition, 15 male partners of these patients were included in the study. HPV-DNA was detected by PCR (polymerase chain reaction) amplification. For HPV-genotyping, PCR products were hybridized with type-specific digoxigenin-labeled oligonucleotide probes and discriminated into 14 high risk (HR) and 6 low risk (LR)-HPV types. The 129 female and 15 male participants were interviewed by a standardized questionnaire for socioeconomic details, drinking, smoking and sexual behaviours. RESULTS: 59 (45.7%) Female participants were negative for a genital HPV-infection. Of these women, 3 (5.1%) showed a positive HPV-PCR result (HR and LR) in the oropharynx. 70 (54.3%) Female participants were positive for a genital HPV infection. In this group, 4 (5.7%) had a positive HPV-detection (HR and LR) in the oral cavity and oropharynx. Female participants with cervical HPV-infection had no higher risk for HPV-detection in the oropharynx (not significant). The analysis of sexual risk factors revealed no specific risk factor for an oral HPV-infection. CONCLUSION: A correlation between cervical and oral colonization by HPV could not be demonstrated in our small cohort. Our limited data suggest that sexual transmission of HPV from the cervix uteri to the oropharynx is a rare and unlikely event.
Assuntos
Alphapapillomavirus/isolamento & purificação , Doenças da Boca/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Feminino , Humanos , Doenças da Boca/epidemiologia , Doenças da Boca/virologia , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologiaRESUMO
Valosin-containing protein (VCP)/p97 has been shown to be associated with antiapoptotic function via activation of the nuclear factor-[Formula: see text]B (NF[Formula: see text]B) signaling pathway and with metastasizing of tumors in several studies. VCP is located on chromosome 9p13-p12, a region often deleted in oropharyngeal squamous cell carcinoma (OSCC). The clinical significance of VCP expression in OSCC however remains unclear. In this study, expression of VCP was determined in 106 patients (77 male (71.3%) and 31 female (28.7%); age-range: 34-79 years (mean age 57 years)) by immunohistochemistry and in a subset of 15 patients by quantitative PCR. HPV-DNA was detected by polymerase chain reaction and p16INK4a immunohistochemistry. The experimental findings were correlated with clinico-pathological data and survival parameters. 47.2% of all OSCC specimens were analyzed as negative or weak staining intensity for VCP. 52.8% of all specimens showed a high staining intensity for VCP. 73.1% of all patients were tested HPV-negative, 26.9% were HPV-positive. The 5-year disease-free and overall survival probabilities of all patients were 71.2% and 55.7%, respectively. No correlation could be found between HPV-status and VCP expression. VCP overexpression in HPV-negative patients was associated with significantly better 5-year disease-free survival (86.4% vs., 45.6%, pâ=â0.017). The level of VCP-intensity determined by immunohistochemistry could be an additional prognostic marker in HPV-negative OSCC. VCP expression seems not to correlate with the HPV-status.
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Adenosina Trifosfatases/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ciclo Celular/metabolismo , Neoplasias Orofaríngeas/diagnóstico , Adenosina Trifosfatases/genética , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ciclo Celular/genética , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/metabolismo , Infecções por Papillomavirus/complicações , Prognóstico , Análise de Sobrevida , Proteína com ValosinaRESUMO
Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16(INK4A) immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.