Associations between oral HPV16 infection and cytopathology: evaluation of an oropharyngeal "pap-test equivalent" in high-risk populations

Human papillomavirus (HPV) is responsible for the rising incidence of oropharyngeal squamous cell cancers (OSCC) in the United States, and yet, no screening strategies have been evaluated. Secondary prevention by means of HPV detection and cervical cytology has led to a decline in cervical cancer incidence in the United States. Here, we explored an analogous strategy by evaluating associations between HPV16 infection, cytopathology, and histopathology in two populations at elevated risk for OSCCs. In the first, a cross-sectional study population (PAP1), cytology specimens were collected by means of brush biopsy from patients presenting with oropharyngeal abnormalities. In the second (PAP2), a nested case-control study, bilateral tonsillar cytology samples were collected at 12-month intervals from HIV-infected individuals. The presence of cytopathologic abnormality in HPV16-positive tonsil brush biopsies (cases) was compared with HPV16-negative samples (controls) matched on age and gender. HPV16 was detected in samples by consensus primer PCR and/or type-specific PCR. Univariate logistic regression was used to evaluate associations. In PAP1, HPV16 alone (OR: 6.1, 95% CI: 1.6-22.7) or in combination with abnormal cytology (OR: 20, 95% CI: 4.2-95.4) was associated with OSCC. In PAP2, 4.7% (72 of 1,524) of tonsillar cytology specimens from HIV-infected individuals without oropharyngeal abnormalities were HPV16 positive. Tonsillar HPV16 infection was not associated with atypical squamous cells of unknown significance (ASCUS), the only cytologic abnormality identified. Therefore, HPV16 was associated with OSCCs among individuals with accessible oropharyngeal lesions but not with cytologic evidence of dysplasia among high-risk individuals without such lesions. An oropharyngeal Pap-test equivalent may not be feasible, likely due to limitations in sampling the relevant tonsillar crypt epithelium.     

Direct uterine sampling with the Tao brush sampler using a liquid-based preparation method for the detection of endometrial cancer and atypical hyperplasia: a feasibility study

BACKGROUND: Endometrial cytology sampling devices for direct uterine sampling have been shown in previous studies to be a reliable and relatively painless method for detecting endometrial lesions. The purpose of the current study was to determine the performance characteristics of endometrial cytology for the detection of malignancy and atypical hyperplasia using liquid-based cytology specimens collected with the Tao brush sampler. METHODS: Brushings of the endometrial cavity were obtained from 139 hysterectomy specimens before routine histopathologic evaluation. Cytology specimens were fixed in PreservCyt and processed using ThinPrep technology. Cytology diagnoses were classified as nondiagnostic, negative, atypical, or positive for malignancy. Histopathologic findings were used as the gold standard for determining the performance characteristics of cytology. RESULTS: Histopathologic results from the 139 patients included 81 (58%) endometrial cancers, 7 (5%) complex hyperplasias with atypia, 2 (1%) complex hyperplasias without atypia, and 49 (35%) patients with benign histology. The number of specimens diagnosed cytologically as positive, atypical, negative, or nondiagnostic was 60 (43%), 40 (29%), 37 (27%), and 2 (1%) specimens, respectively. The overall sensitivity and specificity of cytology for detecting endometrial cancer and atypical hyperplasia were 95% and 66% when atypical cytology specimens were considered positive. CONCLUSIONS: The results of the current study indicate that direct endometrial sampling by liquid-based endometrial cytology collected with the Tao brush sampler produces specimens that contain cellular material that may be identified as endometrial cancer or atypical hyperplasia. Both atypical and positive cytology diagnoses are indicators for triage to more specific methods of diagnosis.     

Oral cancer: Premalignant conditions and screening--an update

Oral cancers form a significant portion of the cancer burden seen in our country. Typically, they tend to be preceded by a premalignant state for a long time. This article discusses the various types of premalignant disorders commonly seen in daily practice. Also, it is important to screen patients for these conditions so as to detect malignant changes early. Previously, the screening of patients for oral cancer and precancerous lesions has relied mainly on conventional oral examination. Nowadays, many newer techniques are available to potentially assist in the screening of healthy patients for evidence of oral cancer. This article attempts to review the current literature for screening methods and adjuncts such as toluidine blue, brush cytology, tissue chemiluminescence and autofluorescence.     

Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers

There are few well-established patient risk factors associated with human papillomavirus (HPV) infection in cancers of the oral cavity and oropharynx. The purpose of this study was to determine if there were significant different risk factors and tumor characteristics between HPV-positive and HPV-negative cancer cases. HPV was evaluated in cancer tissue and exfoliated oral cells of 193 oral cavity/oropharynx cancer patients using PCR and direct DNA sequencing. A patient questionnaire collected information about risk factors, sexual practices and medical history. The prevalence of HPV high-risk (HR) types was 20% in cancer cases. Three types were identified: HPV-16 (87%), HPV-18 (3%) and HPV-33 (11%). Risk factors for HPV-HR included younger age (< or = 55 years vs. > 55 years; adjusted OR = 3.4; 95% CI = 1.6-7.3) and younger-age cases who had more lifetime sex partners (adjusted OR = 3.8; 95% CI = 1.4-10.1), practiced oral-genital sex (adjusted OR = 4.3; 95% CI = 1.8-10.4) or oral-anal sex (adjusted OR = 19.5; 95% CI = 3.4-113). Compared to HPV-negative cancers, HPV-HR cancers were more likely to have a positive HPV-HR exfoliated oral cytology test (adjusted OR = 7.8; 95% CI = 3.4-18.4), later stage (adjusted OR = 3.0), nodal involvement (adjusted OR = 4.1) and advanced grade (adjusted OR = 3.0). This study shows new evidence that the prevalence of oncogenic mucosal HPV is higher in younger-age oral cavity/oropharynx cancer cases whose sexual practices are typically associated with sexual transmission of the virus. HPV detection also appears to be an indicator of advanced disease characteristics that may require different clinical treatment for this subset of patients. An exfoliated oral cytology test for HPV was a significant predictor of HR types in the cancers, suggesting that an oral rinse may provide an early biomarker of infected tumors.     

基于纤支镜刷片细胞学的肺癌细胞核形态的多参数定量研究

目的探讨基于纤维支气管镜刷片细胞涂片中,多参数的细胞核形态定量分析对肺癌的鉴别价值。方法采用回顾性的分析方法,利用HMIAS-2000医学图文分析测量系统,对组织学确诊的纤维支气管镜刷片的细胞核进行形态定量研究(腺癌48例、鳞癌28例、小细胞癌22例,阴性对照组40例)。结果在22个形态定量参数中,19项参数有显著统计学差异(P<0.01),2项有统计学差异(P<0.05),1项无统计学差异(P>0.05)。结论多参数的形态计量揭示了纤支镜刷片细胞学的肺癌与肺良性病变细胞核形态学计量特征,对辅助鉴别诊断良恶性具有一定的意义。     

Analysis of RNA from brush cytology detects changes in B2M, CYP1B1 and KRT17 levels with OSCC in tobacco users

RNA expression analysis of oral keratinocytes can be used to detect early oral cancer, but a limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner, they have not been validated for quantitative analysis of RNA expression. Earlier we showed RNA from brush cytology of hamster Oral Squamous Cell Carcinoma (OSCC) demonstrated differential expression of B2M and CYP1B1 using real time RT-PCR in a dibenz[a,I]pyrene, tobacco carcinogen, induced model of this disease. Here we show reproducibility of this approach to measuring gene expression in humans. Cytology brush samples from 12 tobacco and betel related OSCC and 17 nonmalignant oral lesions revealed B2M mRNA was enriched in tumor samples while CYP1B1 mRNA was reduced, similar to what was seen in the model system. Additionally, we showed that KRT17 mRNA, a gene linked to OSCC in another brush cytology study, was also enriched in OSCC versus nonmalignant lesions, again supporting the promise of using RNA from brush oral cytology to reproducibly monitor oral gene expression.     

Immunocytochemical identification of VPAC1, VPAC2, and PAC1 receptors in normal and neoplastic human tissues with subtype-specific antibodies.

Human tumors frequently overexpress receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). However, none of the VIP/PACAP receptor proteins has been visualized individually in human tumors. Here, we developed and characterized a panel of antipeptide antibodies to the carboxyl-terminal regions of the VIP/PACAP receptor subtypes vasoactive intestinal peptide receptor (VPAC)1, VPAC2, and pituitary adenylate cyclase-activating peptide receptor (PAC)1. Specificity of the antisera was shown by the following: (1) detection of broad bands migrating at Mr 50,000 to 70,000 in Western blots of membranes from receptor-expressing tumors and receptor-transfected cells; (2) cell surface staining of VIP/PACAP receptor-transfected cells; (3) translocation of VIP/PACAP receptor immunostaining in transfected cells after agonist exposure; and (4) abolition of tissue immunostaining by preadsorbtion of the antibodies with their immunizing peptides. The distribution of VIP/PACAP receptors was investigated in 98 human tumors and their tissues of origin. VPAC1, VPAC2, and PAC1 receptors were clearly located at the plasma membrane of the tumor cells in a variety of human neoplasms. In the gastrointestinal tract, VPAC1 receptor immunoreactivity was abundant in the mucosa and myenteric neurons; VPAC2 receptor immunoreactivity was detected in neuroendocrine cells, blood vessels, and smooth muscle; and PAC1 receptor immunoreactivity was found in myenteric neurons. This is the first localization of all of the VIP/PACAP receptor subtypes in human formalin-fixed, paraffin-embedded tissues. VIP/PACAP receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention.     

Differential diagnosis of chronic pancreatitis and pancreatic cancer in brush cytology specimens

Discrimination between chronic pancreatitis and pancreatic carcinoma can be complicated, particularly in brush cytology specimens. Previous studies have shown that the oxygen insensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase activity based on neotetrazolium reduction can be used for discriminating malignant cells from nonmalignant cells. In the present study, we investigated the value of the assay for differential diagnosis between the two pancreatic diseases. Oxygen insensitivity in ductal epithelial cells in normal human pancreas, chronic pancreatitis, and pancreatic carcinoma was determined by quantitative image analysis in sections of biopsies and in brush cytology preparations. In sections, the reaction in the absence of oxygen was a proper reflection of glucose-6-phosphate dehydrogenase activity, whereas in the presence of oxygen only malignant cells showed a significant reaction. Of 39 brush cytology specimens, diagnosis of all 11 cases of pancreatitis and 28 cases of cancer with the oxygen insensitivity test were in agreement with independent measures of chronic pancreatitis and cancer. The oxygen insensitivity test is a simple and valuable tool in addition to conventional pathology for differential diagnosis between pancreatitis and pancreatic cancer, both in biopsies and in brush cytology specimens.     

Usefulness of ultrathin bronchoscopy in diagnosis of lung cancer

Although ultrathin bronchoscopes are suggested to have comparable abilities to conventional bronchoscopes in diagnosing peripheral lung lesions, how to introduce ultrathin bronchoscopes into bronchoscopic examination is still to be determined. In our first study, 35 patients with peripheral lung lesions underwent ultrathin followed by conventional bronchoscopy to compare their diagnostic abilities. The diagnostic rate was 54.3% in conventional bronchoscopy alone, 60.0% in ultrathin bronchoscopy alone, and 62.8% in the combination of the two. In the next study, we introduced a rapid cytology test of the material obtained in conventional bronchoscopy. When malignant cells were not detected in the material by the rapid cytology, ultrathin bronchoscopy was immediately conducted. Thirty-two patients with negative rapid cytology were enrolled in this study. Ultrathin bronchoscopy resulted in diagnostic materials in 59.3% of these cases. Ultrathin bronchoscopes showed better access to the lesions than a brush or a curette introduced through conventional bronchoscopes. We conclude that ultrathin bronchoscopes have a comparable ability to conventional ones in diagnosing peripheral lung cancer even when used alone, and become a good complement to conventional ones by introducing the rapid cytology test.     

Monoclonal antibody and cytological detection of free malignant cells in the peritoneal cavity during resection of colorectal cancer--can monoclonal antibodies do better?

Detection of free malignant cells in the peritoneal cavity following curative resections of colorectal cancer may explain why some patients develop local or peritoneal recurrence after favourable operations. We have examined the incidence of peritoneal malignant cells using standard cytological methods and by indirect immunoperoxidase staining using monoclonal antibodies (CEA L11/285/14 and HMFG 1 and 2) in 30 patients having resection for colorectal cancer. Peritoneal washings were collected on opening the peritoneal cavity and immediately prior to closing the abdominal wall following resection. Abnormal cells were only demonstrated in 10 patients. Cytology revealed abnormal cells in seven patients (three preresection and three postresection, one patient had pre- and postresection positive cytology). Monoclonal antibody staining revealed abnormal cells in seven patients (two preresection and four postresection, one patient had both pre- and postresection positive stains). Only two patients had identical results using cytology and antibody staining. Seven of these 10 patients had hepatic metastases. The correlation between the assessment of free malignant cells using cytology and monoclonal antibody staining is poor. Long-term follow-up is required to see if 'free cells' have prognostic significance.     

Evaluation of surgical excision of non-homogeneous oral leukoplakia in a screening intervention trial, Kerala, India

It is well established that most invasive oral cancers arise from precancerous lesions such as leukoplakia, erythroplakia and oral submucous fibrosis. One of the approaches for control of oral cancer is to detect oral precancerous lesions early in their development and prevent their malignant transformation to invasive cancer either by chemoprevention or by surgical excision of the lesions, with concurrent control of tobacco and alcohol use and other specific aetiological factors. However, the value of specific approaches such surgery in long-term control of lesions and prevention of malignant transformation is not known. We describe our experience with cold knife surgical excision of 59 cases of non-homogeneous leukoplakia of the oral cavity diagnosed in the context of a community-based oral cancer cluster randomised oral cancer screening trial in Kerala, India. Two-thirds of these revealed dysplasia on histology. After a minimum follow-up of 12 months (range 12-37 months) after surgical excision, 44 (74.8%) were remaining disease free with no evidence of recurrent/new lesions; during follow-up, three (5%) developed new luekoplakic lesions, and six (10.1%) developed recurrent lesions, while six (10.1%) could not be traced after treatment. There was no event of malignant change during follow-up. The proportion of subjects remaining with no evidence of disease at 3 years by Kaplan-Meier method of analysis was 62.1% (95% CI: 0.36-0.87). Accrual and long-term follow-up of large number of surgically treated cases may provide valuable leads to management policies of oral leukoplakia, since, as of now, the added value of specific treatments over and above primary prevention by tobacco and alcohol control remains to be established.     

Brush cytology in the diagnosis of colonic neoplasms

During a three-year period (1986-1988), 234 colonic brush specimens were received in the authors' laboratory. Nine samples (4%) were deemed unsatisfactory for evaluation because of inadequate cellularity and/or poor fixation. In 11 cases concomitant or follow-up histologic specimens were not available. The remaining 214 specimens included 82 malignant neoplasms, 88 neoplastic polyps (adenomas), and 44 nonneoplastic lesions. Sixty-seven (82%) of malignant neoplasms were correctly diagnosed by brush cytology. Three cases of adenoma with severe dysplasia or in situ carcinoma were diagnosed as adenocarcinoma by cytology. No false-positive diagnoses were made of nonneoplastic lesions. Brush cytology was found to be a more sensitive technique in the diagnosis of colon cancer than endoscopic biopsy (82% and 74% sensitivity, respectively). The combination of the two techniques increased the sensitivity to 90% and improved the overall accuracy of the test. Seventy-one (82%) of the colonic adenomas were correctly diagnosed by cytology. Brush cytology is a convenient, safe, and accurate technique which should be used concurrently with endoscopic biopsy or polypectomy.     

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.     

Direct uterine sampling with the Tao brush sampler using a liquid‐based preparation method for the detection of endometrial cancer and atypical hyperplasia

BACKGROUND.

Endometrial cytology sampling devices for direct uterine sampling have been shown in previous studies to be a reliable and relatively painless method for detecting endometrial lesions. The purpose of the current study was to determine the performance characteristics of endometrial cytology for the detection of malignancy and atypical hyperplasia using liquid‐based cytology specimens collected with the Tao brush sampler.

METHODS.

Brushings of the endometrial cavity were obtained from 139 hysterectomy specimens before routine histopathologic evaluation. Cytology specimens were fixed in PreservCyt and processed using ThinPrep technology. Cytology diagnoses were classified as nondiagnostic, negative, atypical, or positive for malignancy. Histopathologic findings were used as the gold standard for determining the performance characteristics of cytology.

RESULTS.

Histopathologic results from the 139 patients included 81 (58%) endometrial cancers, 7 (5%) complex hyperplasias with atypia, 2 (1%) complex hyperplasias without atypia, and 49 (35%) patients with benign histology. The number of specimens diagnosed cytologically as positive, atypical, negative, or nondiagnostic was 60 (43%), 40 (29%), 37 (27%), and 2 (1%) specimens, respectively. The overall sensitivity and specificity of cytology for detecting endometrial cancer and atypical hyperplasia were 95% and 66% when atypical cytology specimens were considered positive.

CONCLUSIONS.

The results of the current study indicate that direct endometrial sampling by liquid‐based endometrial cytology collected with the Tao brush sampler produces specimens that contain cellular material that may be identified as endometrial cancer or atypical hyperplasia. Both atypical and positive cytology diagnoses are indicators for triage to more specific methods of diagnosis. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society     

Reverse smoking as a risk factor for palatal cancer: A cross-sectional study in rural andhra pradesh,india

A cross-sectional study of reverse smoking and its association with pre-malignant and malignant lesions of the palate was conducted in the north coastal areas of Andhra Pradesh, India. A total of 480 randomly selected persons were interviewed. Information about smoking status, diet and access to mass media was obtained in each case and an examination of the oral cavity was performed. Reverse smoking of chutta was practised by 33% of the total rural population. The prevalence rate of all palatal lesions was 55%. The prevalence rates of the separate lesions: leukoplakia palatii, palatal keratosis and palatal cancer, were 9.8%, 18.1% and 1.9%, respectively. The presence of these (pre-)malignant lesions was strongly associated with reverse smoking and also associated with conventional chutta smoking. Reverse smoking induced significantly more lesions than conventional chutta smoking, and was a major determinant of subsequent palatal cancer: all 9 newly diagnosed palatal cancers were observed within the group of reverse smokers. There was an inverse relationship between the incidence of palatal lesions and vitamin A intake. The study of access to mass media indicated that the most favourable medium for promoting a prevention campaign would be the cinema.     

Premalignant and malignant cells in sputum from lung cancer patients

BACKGROUND:

The objective of this study was to assess the frequency of premalignant and malignant cells in sputum from patients with lung cancer and to measure the dependence of these cells on cancer stage, histologic type, tumor size, and tumor location.

METHODS:

This analysis included 444 patients with lung cancer. First, all patients were asked to produce sputum spontaneously; then, they underwent sputum induction. Slide preparations of the sputa were screened for the presence of abnormal cells.

RESULTS:

Of all patients with lung cancer who had produced adequate specimens, 74.6% had sputum that was positive for premalignant or worse cells, whereas 48.7% had sputum that was positive for malignant cells alone. Surprisingly, the presence of premalignant or worse cells in sputum depended only moderately on disease stage (82.9% of stage IV cancers vs 65.9% of stage I cancers), tumor size (78.6% of tumors >2 cm vs 64.7% of tumors ≤2 cm), and location (83.3% of central lesions vs 68% of peripheral lesions) and was found to be independent of histologic tumor type (78.4% of squamous cell carcinomas vs 71.5% of adenocarcinomas, 74.5% of small cell carcinomas, and 75% of large cell carcinomas).

CONCLUSIONS:

The findings of the current study suggested the important potential of sputum cytology for lung cancer detection and risk assessment across all stages, histologic types, tumor sizes, and locations. However, the high sensitivities in this study were achieved with a level of scrutiny not feasible in the laboratory routine. The diagnostic potential of sputum cytology may be exploited better through the standardization and automation of sputum preparation and analysis. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Heterogeneous nuclear ribonucleoprotein B1 as early cancer biomarker for occult cancer of human lungs and bronchial dysplasia

Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is a RNA-binding protein of Mr 37,000. We previously reported that hnRNP B1 was specifically overexpressed in the nuclei of human lung cancer cells, particularly in squamous cell carcinoma (E. Sueoka et al., Cancer Res., 59: 1404-1407, 1999). We extended this study to determine whether hnRNP BL was overexpressed in roentgenographically occult cancers of the lungs and premalignant lesions of squamous cell carcinomas, such as bronchial dysplasia. The additional object of our study was to examine the usefulness of hnRNP B1 as a potential diagnostic marker for squamous cell carcinoma of various organs, such as the oral cavity and esophagus in humans. Surgically resected specimens of bronchial dysplasia, lung cancers, and various human squamous cell carcinomas, collected at two hospitals in Japan, were subjected to immunohistochemical staining with anti-hnRNP B1 antibody. Overexpression of hnRNP B1 protein was observed in 100% of stage I lung cancer tissues, but it was not found in normal bronchial epithelium. Squamous cell carcinoma of the lungs showed stronger staining than other histological types, and elevation of hnRNP B1 was found in both roentgenographically occult lung cancers and bronchial dysplasia. Furthermore, cytological examination with anti-hnRNP B1 antibody detected cancer cells in sputum, suggesting the potential of hnRNP B1 protein as a new biomarker for the very early stage of lung cancer in humans. Because strong staining of hnRNP B1 was also observed in various squamous cell carcinomas of oral and esophageal tissues as shown in our recent reports, overexpression of hnRNP B1 seems to be a common event in the carcinogenic processes of squamous cell carcinoma. These results suggest that hnRNP B1 protein could be a useful diagnostic biomarker for both the very early stages of lung cancer and various squamous cell carcinomas in humans.     

Biliary brush cytology in the assessment of biliary strictures at a tertiary center in Iran

Background: Confirmation of cholangiocarcinoma and other malignant bile duct stenosis is challenging. The aim of the current study was to assess the accuracy of brush cytology for diagnosis of malignant biliary strictures. Methods: 105 patients with hepatic biliary strictures undergoing ERCP were included in this study. Prospectively collected data included symptoms, results of biochemical testing and imaging procedures, as well as details of ERCP. Exclusion criteria were: 1) strictures that would not permit passage of guidewire and brush accession; and 2) post-operative strictures. Brushings of the bile duct strictures were performed. All patients were followed for at least 6 months. The final diagnosis was confirmed following surgery, histopathological diagnosis of the lesion, radiological infiltration of adjacent organs or metastases, or after at least a 6-month follow-up. Results: 88 brush samples from 88 patients were of appropriate quality. The overall diagnostic sensitivity and specificity for malignant nature of biliary strictures were 40.7% and 100%, respectively. The sensitivity was 66.6 % for ampullary carcinomas, 36.3% for pancreatic cancer and 32.5% for cholangiocarcinomas. Conclusions: Despite the low sensitivity, due to the relative ease and safety, brush cytology should remain the first choice for diagnosis of causes of biliary strictures.