Absence of human papillomavirus in tonsillar squamous cell carcinomas from Chinese patients

Epidemiological and experimental evidence from Western countries now consistently support an etiological role for human papillomavirus (HPV) in a subset of oropharyngeal squamous cell carcinomas (SCC), especially those originating in the tonsil. The role of HPV in the etiology of tonsil cancer in developing countries such as China has not been investigated. In this study, none of 16 tonsil cancer specimens from Chinese patients were positive for HPV DNA, whereas those from Australian patients using the same methodology gave a positivity rate of 46%. The tumors from Chinese patients, like the Australian HPV-negative subset, significantly overexpressed pRb and cyclin D1 and underexpressed p16(INK4A) (p16). In contrast, the Australian HPV-positive cancers overexpressed p16 and had reduced expression of pRb and cyclin D1. These findings may help explain why China has a relatively low rate of oropharyngeal cancer compared with Australia. They also support the hypothesis that molecular pathways to tonsil cancer mediated by HPV are distinct from those induced by mutagens present in cigarette smoke or alcohol.     

Human papillomavirus in squamous cell carcinoma of the oesophagus associated with tylosis.

Human papillomavirus (HPV) may have a pathogenic role in squamous cell carcinoma of the oesophagus. Tylosis, an inherited thickening of the skin of the palms and soles, was associated with a high risk of developing squamous cell carcinoma of the oesophagus among members of a large family in Liverpool. The resected carcinomas of the oesophagus was examined from four such patients with DNA probes to HPV types 6,11,16,18,31,33 and 35 using in situ hybridisation under conditions of high stringency. No reaction was detected. The oesophageal biopsy specimens from 10 tylotic subjects without carcinoma were also examined. No HPV DNA was detected. It is concluded that there is no evidence that HPV infection has a role in the development of squamous cell carcinoma of the oesophagus in tylosis.     

Human papillomavirus DNA in squamous cell carcinoma of the lung.

AIM: To compare the incidence of squamous cell carcinoma (SCC) of the lung in Okinawa with that in Niigata on the mainland. METHODS: All patients presenting with SCC of the lung in Okinawa and Niigata in 1993 were included in the study. Diagnoses were confirmed by conventional histological examination of paraffin wax sections. Human papillomavirus (HPV) was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for the E6 and E7 regions of the HPV genome. PCR products were analysed by Southern and dot blotting. RESULTS: The incidence of well differentiated SCC of the lung was high in patients from Okinawa compared with moderately and poorly differentiated types, and compared with the incidence of SCC in patients from Niigata. This is despite similar patterns of age, sex (predominatly male), and smoking habit. More patients from Okinawa, however, were positive for HPV DNA by PCR (79%) and NISH (53%). Many patients haboured HPV types 6, 16, and 18. Only 30% of patients from Niigata were positive for HPV DNA by PCR and 20% by NISH. These patients all harboured one HPV type only. CONCLUSION: Surprisingly large numbers of patients from Okinawa were positive for HPV DNA. The detection of HPV DNA was strongly associated with well differentiated SCC. This was particularly true for HPV types 6 and 16. There was no correlation between either smoking and detection of HPV DNA, or smoking and histological differentiation.     

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.     

Detection of mucosal type human papillomavirus in cutaneous squamous cell carcinoma in Iran

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer. The oncogenic role of human papilloma virus (HPV) in cutaneous SCC has been suggested by several studies performed on immunosuppresed patients. However, the role of mucosal type HPV in SCC patients with normal immunity has not been studied extensively. Sixty skin biopsies from immunocompetent SCC patients and 60 benign skin specimens were evaluated for mucosal type HPV DNA using polymerase chain reaction (PCR) and immunohistochemistry (IHC). Mucosal type HPV DNA was detected in 18 of 60 cases (30%) and in 7 of 60 controls (11.6%) using PCR. HPV immunostain was positive in 16 of 60 cases (26.6%) and in 15 of 60 controls (25%). Mixed infection with HPV 18, 11, 6 was found in half of the SCC cases. The most prevalent subtype was HPV 18 followed by HPV 6 and 11. The frequency of HPV DNA was significantly elevated in our cases compared to controls (P value <0.01, OR = 16.8, 95% CI: 3.3–74.9). Our findings suggest an association of mucosal type HPV, especially HPV 18, with skin SCC in Iranian patients with normal immunity.     

The biomarkers of human papillomavirus infection in tonsillar squamous cell carcinoma-molecular basis and predicting favorable outcome.

Presence of human papillomavirus (HPV) in variable proportions in tonsillar squamous cell carcinoma tissues has been demonstrated by several worldwide studies. Some reports emphasized the significance of HPV in predicting a better prognosis, as well as ethnic differences between Chinese and Caucasians. In order to understand the biological role of HPV and find out clinically accessible methods to determine its prognostic significance in primary tonsillar squamous cell carcinoma, we collected 92 patients with primary tonsillar squamous cell carcinoma diagnosed or treated in National Taiwan University Hospital, for whom archival tumor tissue were available. Immunohistochemical stains of p16(INK4A), high-risk HPV in situ hybridization, and nested polymerase chain reaction (PCR)-based genechips were performed to detect HPV infection and determine its genotype. Clinical data were compared with HPV infection detected by the different methods mentioned above. Real-time PCR was also performed on the HPV16-positive [HPV16(+)] lesions to understand viral integration status. The positive rates of nested PCR-based genechips, overexpression of p16(INK4A), and high-risk HPV in situ hybridization were 75% (69/92), 53% (49/92), and 44% (40/92), respectively. Both overexpression of P16(INK4A) and high-risk HPV in situ hybridization positivity were associated with favorable prognoses (P=0.004 and 0.001, respectively) and also independent prognostic factors in multivariate analyses (P=0.01 and 0.01, respectively). The positivity of nested PCR-based genechips was not statistically significant. From our data, primary tonsillar squamous cell carcinoma with positive immunohistochemical stains of p16(INK4A) and/or high-risk HPV in situ hybridization is associated with a better outcome, and both methods may serve as clinically accessible markers.     

Association between human papillomavirus infection and laryngeal squamous cell carcinoma

The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR‐DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin‐embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti‐human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF₁₀ PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma. J. Med. Virol. 82:1017–1023, 2010. © 2010 Wiley‐Liss, Inc.     

Human papillomavirus in oesophageal squamous cell carcinoma.

Thirty seven cases of oesophageal squamous cell carcinoma were studied by applying DNA slot blot analysis and in situ hybridisation using type specific probes for HPV 6, 11, 16 and 18. Cases of condyloma accuminata, cervical carcinoma, and laryngeal papilloma were used as controls. Blocks including areas of invasive carcinoma, intraepithelial neoplasia, and normal epithelium were studied in each case. No HPV genome was detectable in any of the oesophageal cases. It is concluded that these types of HPV do not have an association with oesophageal squamous cell carcinoma.     

Human papillomavirus infection is not associated with laryngeal squamous cell carcinoma in Taiwan

Background/PurposeTo examine whether the prevalence rate of human papillomavirus (HPV) infection in Taiwanese patients with primary laryngeal squamous cell carcinoma (LSCC) is different from that in those with a vocal polyp (VP) or vocal fold leukoplakia (VFL).MethodsThis prospective cohort study recruited 41 consecutive patients with primary LSCC and 27 and 20 patients with VP and VFL, respectively. The HPV L1 gene in surgical specimens was detected using polymerase chain reaction. High-risk HPV DNA in tissue microarray specimens was detected using in situ hybridization. Expression of p16^INK4a in tissue microarray specimens was determined through immunohistochemistry.ResultsThe prevalence of HPV L1 DNA in the LSCC group was equivalent to that in the VP and VFL groups (7.3% vs. 7.4% vs. 10.0%; P = 0.929; effect size = 0.20). High-risk HPV DNA detected using in situ hybridization was relatively rare in all groups (2.6% vs. 5.3% vs. 0.0%; P = 0.636; effect size = 0.81). The prevalence of p16^INK4a positivity was significantly lower in the LSCC group than in the VP and VFL groups (5.1% vs. 58.8% vs. 14.3%; P < 0.001). Multivariate analysis results revealed that age ≥65 years (adjusted odds ratio, 4.09; 95% confidence interval, 1.21–13.91; P = 0.024) and p16^INK4a positivity (adjusted odds ratio, 0.10; 95% confidence interval, 0.02–0.53; P = 0.006) were LSCC risk factors.ConclusionHPV infection is uncommon in Taiwanese patients with LSCC and seems not to be associated with an increased LSCC risk. Larger sample size is warranted for further study.     

Human papillomavirus type 11DNA in papillary squamous cell lung carcinoma

Summary We report a case of papillary squamous cell carcinoma of the lung developing in relation to a condylomatous papilloma and related to human papillomavirus (HPV) infection. The viral origin of the bronchial papillomatous lesion is strongly suggested by cytological and histological features with marked condylomatous changes. No viral capsid antigen was detected by immunohistochemistry. Transmission electron microscopy failed to reveal intranuclear viral-like particles in the papillary part of the carcinoma, but typical ultrastructural koilocytotic cells with irregular nucleus and coarse chromatin were observed. HPV DNA type 11 was detected by in situ hybridization using biotinylated probes on paraffin-embedded specimens, under stringent conditions (T _m }-12, 50% formamide). Papillary squamous cell carcinoma may result from the malignant conversion of benign squamous papilloma of the bronchus. HPV type 11 may be associated with malignant conversion of benign papilloma of the pulmonary tract, as in the upper respiratory tract. In situ hybridization with biotinylated probes is a relatively simple and appropriate method for retrospective analysis of HPV DNA sequences in surgical specimens.     

Basic emotions are associated with distinct patterns of cardiorespiratory activity.

The existence of specific somatic states associated with different emotions remains controversial. In this study, we investigated the profile of cardiorespiratory activity during the experience of fear, anger, sadness and happiness. ECG and respiratory activity was recorded in 43 healthy volunteers during the recall and experiential reliving of one or two potent emotional autobiographical episodes and a neutral episode. Univariate statistics indicated that the four emotions differed from each other and from the neutral control condition on several linear and spectral indices of cardiorespiratory activity. Dependent variables were further reduced to five physiologically meaningful factors using an exploratory principal component analysis (PCA). Multivariate analyses of variance and effect size estimates calculated on those factors confirmed the differences between the four emotion conditions. A stepwise discriminant analyses predicting emotions using the PCA factors led to a classification rate of 65.3% for the four emotions (chance=25%; p=0.001) and of 72.0-83.3% for pair-wise discrimination (chance=50%; p's<0.05). These findings may be considered preliminary in view of the small sample on which the multivariate approach has been applied. However, this study emphasizes the need to better characterize the multidimensional factors involved in cardio-respiratory regulation during emotion. These results are consistent with the notion that distinct patterns of peripheral physiological activity are associated with different emotions.     

Detection of human papillomavirus using hybrid capture 2 in oral brushings from patients with oropharyngeal squamous cell carcinoma

Detection of high-risk (HR) human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (SCC) has important prognostic implications; patients exhibit improved survival compared with patients with HPV- SCC. Oral brushing and rinsing samples were obtained from patients with oropharyngeal, oral cavity, or hypopharyngeal SCC and tested for HR-HPV using Hybrid Capture 2 (HC2; QIAGEN, Valencia, CA). HR-HPV in situ hybridization (ISH) was performed on biopsy tissue samples from the same patients. Oral cytologic samples from 16 SCCs were tested by HC2. Biopsy tissue samples were available for ISH in 11 cases. Five oropharyngeal SCCs were HR-HPV+ by ISH and HC2 (oral brushing). Of the oropharyngeal SCCs, 2 were positive by HC2 (oral brushing) and negative or equivocal by ISH. We found that 2 oral cavity carcinomas and 2 hypopharyngeal carcinomas were negative by HC2. One hypopharyngeal cancer was positive by ISH. All oral rinsing samples were negative by HC2. HC2 may be an effective method of determining HR-HPV status in patients with oropharyngeal SCC.     

Overexpression of phosphorylated nuclear factor-kappa B in tonsillar squamous cell carcinoma and high-grade dysplasia is associated with poor prognosis.

Intracellular signals along the epidermal growth factor receptor (EGFR)-Akt-nuclear factor-kappa B (NF-kappaB) pathway have been associated with carcinogenesis in various malignant neoplasms. This investigation was to evaluate the expression of EGFR, phosphorylated(p)-Akt and p-NF-kappaB and correlate them with clinical outcomes in patients with squamous cell carcinoma of the tonsil. A total of 45 patients with squamous cell carcinoma of the tonsil were studied by immunohistochemistry to evaluate the expression levels of EGFR, p-Akt and p-NF-kappaB. Results for squamous cell carcinoma of the tonsil were compared with those for associated high-grade dysplasia and adjacent normal appearing epithelium, when present. In addition, tonsillar epithelium from non-neoplastic specimens of age-matched patients also was stained for the same markers. High-grade dysplasia and squamous cell carcinoma of the tonsil demonstrated a similar pattern of expression, which differed from the pattern seen in the adjacent normal epithelium and tonsillar epithelium from normal controls (an overexpression for each of these three protein analytes in high-grade dysplasia and squamous cell carcinoma of the tonsil as demonstrated by immunohistochemistry). When markers from squamous cell carcinoma of the tonsil were correlated with survival status, only increasing levels of p-NF-kappaB immunoreactivity (a relative overexpression) were statistically significant predictors of poor survival. No markers in squamous cell carcinoma of the tonsil were significantly related to rate of recurrence. When analyzing marker scores from tissue with high-grade dysplasia, relative overexpressions of both p-Akt and p-NF-kappaB were significantly related to poor survival. Additionally, increasing levels of p-NF-kappaB immunopositivity from tissue with high-grade dysplasia were also significantly related to rate of recurrence. In summary, p-NF-kappaB, overexpressed in high-grade dysplasia and squamous cell carcinoma of the tonsil, is associated with worse prognosis in terms of high recurrence and poor survival, respectively. This significant finding in patients with squamous cell carcinoma of the tonsil, in combination with previous animal and in vitro studies, suggests that p-NF-kappaB represents a potential therapeutic target in head and neck squamous cell carcinoma.     

喉鳞状细胞癌患者人乳头瘤病毒感染的检测分析

目的 了解喉鳞状细胞癌患者人乳头瘤病毒感染的情况.方法 对收集到的64例临床诊断喉癌病例的石蜡组织标本应用Luminex及PCR的方法对HPV感染进行基因检测,应用免疫组织化学的方法对HPV16/18E6蛋白进行检测.结果 在64例临床诊断的喉癌病例中,通过Luminex及PCR的方法发现7例病例具有HPV的感染;通过免疫组织化学的方法发现18例病例具有HPV16/18的感染,基因及蛋白检测的总阳性率达到39.1％.结论 此研究发现在喉癌患者中较高的HPV感染率间接说明HPV感染对喉癌发生的重要性,为阐明喉癌的发病机制奠定基础.     

Detection of human cytomegalovirus DNA in human tonsillar lymphocytes

The human cytomegalovirus (HCMV) was first isolated in cell cultures from the oropharynx, which is thought to be a site of primary infection. Although HCMV can be recovered from the oropharynx during reactivation phases, its exact site of latency is not known. In the present study we demonstrated evidence suggesting the presence of latent HCMV in this anatomic region--in the palatine tonsils. Samples from 30 tonsils obtained by tonsillectomy were screened for the presence of HCMV. Out of the 30 tonsil donors, 23 were seropositive for HCMV. Three methods were used in attempts to demonstrate HCMV's presence in the tonsils: (1) viral isolation attempts on various cell cultures, (2) immunohistochemical staining--immunoperoxidase method--designed to detect viral antigens, and (3) DNA dot hybridization with a HCMV-DNA probe designed to detect viral DNA. Neither infectious HCMV nor other viruses were isolated in cell cultures. No viral antigens were detected by immunoperoxidase staining in the tonsillar tissue. Four out of the 30 tonsils studied were found to contain viral DNA. In one case in which the tonsillar mononuclear (MN) fraction was separated from the polymorphonuclear (PMN) fraction, only the first fraction contained the viral DNA.     

基因芯片检测人肺鳞癌和肺腺癌基因表达的异同

目的用基因芯片技术检测肺鳞癌和肺腺癌基因表达的异同。方法提取人肺鳞癌和肺腺癌组织及正常肺组织的RNA,分别用Cy5-dCTP或Cy3-dCTP标记,再与4096点基因芯片杂交,检测肺鳞癌和肺腺癌组织基因表达的异同。结果肺鳞癌和肺腺癌表达共同上调的基因17条,共同下调的基因19条;肺鳞癌表达显著高于肺腺癌的基因20条,显著低于肺腺癌的基因14条。结论多基因参与肺癌发病,基因芯片技术是肺癌基因表达检测的有效方法。     

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2-induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV-negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low-grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non-neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.