Cytologic diagnosis of Ewing sarcoma/peripheral neuroectodermal tumor with paired prospective molecular genetic analysis.

BACKGROUND: Ewing sarcoma/peripheral neuroectodermal tumor (EWS/PNET), since its characterization immunophenotypically and cytogenetically, has emerged as one of most common sarcomas of childhood. Currently, it is recognized that EWS/PNET can occur in any number of extraosseous sites and is one of several distinctive tumor types with an EWS translocation. In the past, the pathologic diagnosis of EWS/PNET relied on an open biopsy with the application of various ancillary studies, ranging from periodic acid-Schiff stain to molecular testing, but the tumor increasingly is diagnosed on the basis of cytologic specimens alone. METHODS: The authors report 3 cases of EWS/PNET in patients aged 11 years to 53 years. These 3 patients had tumors that involved the parotid gland, cervical soft tissue, and pelvis, and were diagnosed by cytologic evaluation of fine-needle aspiration (FNA) biopsy material alone. The FNA materials also were evaluated prospectively by fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction. RESULTS: The results emphasized the diagnostic utility of FNA biopsy material for morphologic and molecular analysis without compromising conventional cytologic and immunocytochemical analysis, and that prospective molecular testing of FNA specimens has utility in routine practice although it is subject to many of the same limitations that impact molecular analysis when applied to conventional tissue biopsy specimens. CONCLUSIONS: The current results demonstrated that molecular genetic techniques can provide clinically useful ancillary information for FNA specimens when cytologic features and/or immunophenotype are equivocal on the basis of limited sampling or secondary changes, such as hemorrhage and/or necrosis.

Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer.

Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer.

Prevalence of high-risk human papillomavirus DNA in nonkeratinizing (cylindrical cell) carcinoma of the sinonasal tract: a distinct clinicopathologic and molecular disease entity.

Carcinomas of the nose and paranasal sinuses are a heterogeneous group of neoplasms that differ histologically, biologically, and clinically. Some of these tumors are known to harbor high-risk human papillomavirus (HPV) DNA. In an attempt to identify specific phenotypes associated with HPV infection, 39 cases of sinonasal carcinomas were evaluated by PCR for the presence of HPV DNA. The tumors were also studied with a panel of immunohistochemical stains, including p16, p53, and Ki-67 antibodies. Twenty-one cases were identified as keratinizing squamous cell carcinoma (KSCC) with a male-to-female ratio of 3:1. Eight cases were nonkeratinizing (cylindrical cell) carcinoma (NKCa) with a male-to-female ratio of 1:1. Ten cases were sinonasal undifferentiated carcinoma (SNUC), and 9 of these patients were men. HPV DNA, particularly type 16, was detected in 9 cases: 4 of 21 (19%) of KSCC, 4 of 8 (50%) of NKCa, and 1 of 10 (10%) of SNUC. In addition to a higher prevalence of HPV DNA in NKCa, the tumors also showed a distinct immunophenotype characterized by strong and diffuse staining for p16, high labeling scores for Ki-67, and negative or low reactivity to p53. On the other hand, KSCC and SNUC were either negative or weakly reactive to p16 antibodies. KSCC cases were more likely to be positive and more strongly reactive to p53 stain. Unlike KSCC, SNUC had high Ki-67 labeling scores. These observations suggest that NKCa of the sinonasal tract is a distinct histopathologic and molecular disease entity, which should be added to the list of upper aerodigestive tract tumors with strong etiologic relationship to high risk HPV.

Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix.

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of primary small cell carcinoma of the uterine cervix, a rare but highly aggressive malignancy. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In squamous cell carcinoma and adenocarcinoma of the cervix, HPV infection is also associated with overexpression of p16, a cyclin-dependent kinase inhibitor. In this study, 22 cases of primary small cell carcinoma of the uterine cervix were subjected to broad-spectrum HPV DNA amplification and typing, and immunohistochemically examined for the expression of p16, Rb, and p53 proteins. The results show that HPV DNA was detected in every case (100%), with 18 cases (82%) harboring type 18. The tumor cells exhibited strong nuclear staining for p16 in 20 cases (91%). This was associated with a complete loss of Rb nuclear staining in tumor cells in 16 cases (73%). The p53 protein was essentially undetectable in all cases. In contrast, HPV DNA was not detected in 9 colorectal and 8 urinary bladder small cell carcinomas included in this study for comparison. While similar p16 and Rb expression patterns were observed in these HPV-negative tumors, a different expression pattern for p53 was noted where strong nuclear staining was seen in 8 cases (47%; P = 0.0004 compared with cervical tumors). These observations indicate that different mechanisms are involved in the pathogenesis of small cell carcinomas of the uterine cervix and support the notion that nuclear p16 overexpression serves as an indication of Rb defunctioning in tumor cells, which may or may not result from high-risk HPV infection.

ThinPrep evaluation of fluid samples aspirated from cystic ovarian masses.

The cytological evaluation of ovarian cystic fluid using ThinPrep has not been reported. To determine the diagnostic accuracy of ThinPrep cytology in distinguishing between benign and nonbenign ovarian cystic lesions, we examined 65 fluid samples aspirated during intraoperative consultation with subsequent histologic correlation. One ThinPrep slide was prepared from each sample aspirated from surgically removed ovarian cystic masses and reviewed blindly by a panel of three cytopathologists. The parameters used in cytological evaluation were cellularity, cell types, cellular arrangement, and background. Four samples were acellular and excluded from the study. The consensus cytologic diagnoses were compiled for 61 cases which were assigned to one of the following diagnostic categories: negative for malignant cells (40 cases), atypical cytology (13 cases), and suspicious or positive for malignancy (8 cases). Histologic correlation of the cytological benign/negative cases showed that 26/40 (65%) were histologically benign and 14/40 were false-negative (35%, 5 carcinomas and 9 borderline tumors) with 10 of these cases being mucinous tumors. Most false-negative cytologic samples (11/14 or 79%) did not have an epithelial component. Of the 21 cytological nonbenign diagnoses (atypical/suspicious/positive), 15 (71%) were confirmed on histology (10 carcinomas and 5 borderline tumors). However, a nonbenign cytologic diagnosis was rendered in 6 histologically benign cases, including 2 serous cystadenomas, 1 mucinous cystadenoma, 1 serous cystadenofibroma, 1 endometriosis, and 1 corpus luteal cyst. The diagnostic sensitivity by ThinPrep evaluation of ovarian cystic masses is 81% (26/32) for benign and 52% (15/29) for nonbenign lesions. Our results concluded that ThinPrep examination of ovarian cystic fluid is not accurate in distinguishing benign from malignant cysts, given the significant number of false-negative diagnoses. Major contributing factors include sparse cellularity of the fluid samples and mucinous differentiation of the tumors.

Double cervix with bilateral and synchronous HSIL associated with different high-risk HPV types. A case report.

BACKGROUND: High grade squamous intraepithelial lesion (HSIL) of the cervix is well known to be associated with human papillomavirus (HPV) infection. HSIL and invasive carcinomas occurring synchronously in genital malformations, such as a double cervix, have been reported. It has been postulated that the field effect phenomenon of HPV infection is responsible for this synchronous infection. However, there is no information in the literature on the specific types of HPV causing the concomitant lesions in cases with a double cervix. CASE: A 33-year-old nulligravida with a double cervix and a single uterine corpus was diagnosed with bilateral HSIL on Papanicolaou-stained ThinPrep slides (Cytyc Corp., Boxborough, Massachusetts, U.S.A.). A bilateral loop electrosurgical excision procedure cone biopsy revealed HSIL involving both cervices. DNA extracted from the HSIL lesions was analyzed by a polymerase chain reaction-based assay for the presence of HPV. High-risk HPV type 33 was identified in the right cervix, while HPV type 35 was present in the left. CONCLUSION: Demonstration of high-risk HPV types bilaterally supported the etiologic role of HPV infection in the synchronous and bilateral occurrence of HSIL in this case of a double cervix. The HPV types were different in the right and left cervices.

Overexpression of c-kit protein is an infrequent event in small cell carcinomas of the uterine cervix.

Primary small cell carcinomas of the uterine cervix are uncommon but highly aggressive malignancies. The recent observations that c-kit proto-oncogene, a tyrosine kinase, is overexpressed in small cell lung cancers and that advanced c-kit-expressing gastrointestinal stromal tumors were successfully treated with a selective tyrosine kinase inhibitor STI-571 (Gleevec, imatinib mesylate) prompted us to investigate c-kit protein expression in cervical small cell carcinomas. Using a polyclonal antibody against c-kit protein (CD117), our immunohistochemical studies demonstrated a cytoplasmic staining in six of 22 cases (27%) of cervical small cell carcinoma. However, in five of these c-kit-positive cases, the immunoreactivity was focal and/or weak. Only one case (5%) exhibited a strong and diffuse staining pattern comparable to that seen in gastrointestinal stromal tumors. This was in contrast to small cell lung cancers where a positive staining for c-kit was observed in nine of 14 cases (64%) included in the study for comparison. Among them, five (36%) exhibited a strong and diffuse staining pattern. These results indicate that overexpression of c-kit protein is an infrequent event in small cell carcinomas of the uterine cervix. In comparison with small cell lung cancers, the findings presented in this report may reflect the difference in etiopathogenetic mechanisms underlying these two types of small cell carcinomas.

Atypical squamous cells of undetermined significance in girls and women.

OBJECTIVE: To estimate the outcome of adolescents with atypical squamous cells of undetermined significance (ASC-US) on cytology. METHODS: A review of ASC-US cytology in girls and women aged 10-19 years between 1995 and 1999 was performed. The cytologic and histologic follow-up of each patient was evaluated. The outcome was recorded as the most significant (highest grade) subsequent cervical smear or biopsy. RESULTS: Overall, 535 of 7897 (6.8%) cervical cytologic specimens were reported as ASC-US. The study group consisted of 398 patients for whom pathologic follow-up was available. The mean duration of follow-up was 19 months. Follow-up consisted of repeat cytology in 251 (63%) patients and colposcopy with cervical biopsies and/or endocervical curettage in 147 (37%) of the adolescents. Two hundred fifty-three (64%) adolescents had no pathologic abnormalities on follow-up. Persistent ASC-US was identified in 65 (16%), low-grade squamous intraepithelial lesions/cervical intraepithelial neoplasia (CIN) 1 was found in 44 (11%) and high-grade squamous intraepithelial lesions/CIN 2 or 3 occurred in 36 (9%) of the adolescents. No cases of invasive carcinoma were found. CONCLUSIONS: Among adolescents with ASC-US, the rate of squamous intraepithelial lesions/CIN is similar to that of adults. Although the optimal management of ASC-US in adolescents is unknown, these patients warrant close follow-up. LEVEL OF EVIDENCE: III

Metachronous squamous cell carcinomas evolving from independent oropharyngeal and pulmonary squamous papillomas: association with human papillomavirus 11 and lack of aberrant p53, Rb, and p16 protein expression.

Squamous cell carcinoma evolving from squamous papilloma in both the upper and lower respiratory tract in the same patient is uncommon. The molecular mechanisms underlying the progression have not been well investigated. We herein describe a case of squamous cell carcinoma arising from respiratory papilloma in two independent occasions. The patient initially had oropharyngeal squamous cell carcinoma arising in a squamous papilloma at the age of 25 years. He subsequently developed squamous cell carcinoma in the left lower lobe of the lung, which was also associated with squamous papilloma, 8 years after the complete excision of the oropharyngeal lesion. Polymerase chain reaction-based broad-spectrum human papillomavirus DNA amplification and typing showed the presence of human papillomavirus type 11 DNA in both oropharyngeal and pulmonary tumors. Immunohistochemical studies showed that the expression status of p53, Rb, and p16 proteins was unaltered during tumor progression. These observations indicate that human papillomavirus 11-associated neoplastic transformation and tumor progression in the respiratory tract may not involve aberrant regulation of the p53 and Rb signaling pathways.

Prevalence and typing of HPV DNA in atypical squamous cells in pregnant women.

OBJECTIVE: To determine the prevalence and typing of HPV DNA in pregnant women with a diagnosis of atypical squamous cells (ASC) and to assess whether pregnancy-related changes contribute to the diagnosis of ASC. STUDY DESIGN: HPV testing was performed on residual specimens from the ThinPrep Pap test (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) in pregnant women diagnosed as ASC (study group, n = 105), low and high grade squamous intraepithelial lesion (LSIL and HSIL) (positive control, n = 33) and negative for epithelial cell abnormality (negative control, n = 20). All cases were reviewed by 2 cytopathologists to obtain consensus diagnoses using the Bethesda System 2001 criteria. The study group cases were further subcategorized into ASC of undetermined significance (ASCUS, n = 99) and ASC cannot exclude HSIL (ASC-H, n = 6). HPV testing was also performed on an ASC control group consisting of 68 consecutive ASC cases in nonpregnant women, matched by age. RESULTS: Mean patient age was 23.7 years for the study group and 25.6 years for the ASC control group. HPV DNA was detected in 88.6% of cases in the study group, including 87.9% of ASC-US and 100% of ASC-H cases. Of the HPV positive cases, 79.6%, 4.3%, 5.4% and 10.8% had high-risk, mixed high- and low-risk, low-risk and unknown HPV types, respectively. The most frequent HPV types detected were: types 52 (31.2%), 16 (15.1%), 39 (11.8%), 53 (10.8%), and 18 and 58 (9.7% each). Multiple viral types were detected in 43.0% of cases. The prevalence of HPV DNA in the positive and negative controls in pregnant women was 100% and 55%, respectively. HPV DNA was detected in 83.8% of the ASC control group. CONCLUSION: Regardless of pregnancy-related changes, the prevalence of HPV DNA in pregnant women (88.6%) was similar to that found in ASC in nonpregnant women of the same reproductive-age group (83.8%), and the high-risk types accounted for the vast majority of cases (83.9%). These findings demonstrate that pregnancy-related changes do not contribute to the diagnosis of ASC in this subset of women. Furthermore, the high HPV DNA prevalence in reproductive-age women (< 40 years) suggests that HPV testing may have limited utility in effective management of these patients.

Prevalence of human papillomavirus type 16 DNA in squamous cell carcinoma of the palatine tonsil, and not the oral cavity, in young patients: a distinct clinicopathologic and molecular disease entity.

Squamous cell carcinoma of the head and neck commonly affects patients in their sixth decade and older, particularly those with a prolonged history of alcohol and tobacco abuse. Less frequently, carcinomas occur in young individuals even in the absence of known risk factors. The purpose of this study is to investigate a possible relationship between these tumors and human papilloma virus (HPV). Thirty-three cases of squamous cell carcinoma of the head and neck in young patients under the age of 40 years were studied: 15 oral, 11 tonsillar, and 7 laryngeal. HPV DNA was detected by polymerase chain reaction in 10 tonsillar and 2 laryngeal carcinomas and in none of the oral tumors. Of the 12 HPV-positive tumors, 11 were HPV16 and 1 was HPV31. HPV-positive tumors had a distinct nonkeratinizing basal cell morphology, they stained diffusely and strongly with p16 antibodies, had higher Ki-67 and lower p53 staining scores as compared with the conventional keratinizing HPV negative carcinomas. It is concluded that in young patients high-risk HPV, particularly HPV16, is strongly associated with tonsillar squamous cell carcinoma and some cases of laryngeal, but not oral, tumors. The HPV-positive carcinomas have a distinct histopathologic and immunophenotypic features.

Expression of p16, Rb, and p53 proteins in squamous cell carcinomas of the anorectal region harboring human papillomavirus DNA.

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of squamous cell carcinoma of the anorectal region. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In cervical and head and neck cancers, HPV infection is also associated with an overexpression of p16, a cyclin-dependent kinase inhibitor. The expression of these cell cycle regulators in squamous cell carcinomas of the anorectal region has not been well studied. In the current study, 29 cases of squamous cell carcinoma of the anorectal region were immunohistochemically examined for the expression of p16, Rb, and p53 proteins. Tumor cell DNA was also extracted from paraffin blocks and subjected to broad-spectrum HPV DNA testing and typing. The results show that the tumor cells exhibited a strong and diffuse nuclear stain (with some cytoplasmic positivity) for p16 in all 29 cases (100%). The adjacent nonneoplastic squamous epithelium or colonic mucosa, in contrast, was completely negative. Loss of Rb nuclear staining in tumor cells was observed in 20 cases (69%). The p53 protein was essentially undetectable, with only 6 cases containing <10% positive cells. HPV DNA was detected in every case (100%), with 25 cases (86%) harboring Type 16. In addition, almost identical results were obtained in 12 HPV-positive squamous cell carcinomas of the upper aerodigestive tract. This was in marked contrast to those of HPV-negative tumors, where positive p16 staining and loss of Rb expression were seen in only 2/21 (10%) and 1/21 (5%) cases, respectively. These observations indicate that overexpression of p16 and loss of Rb nuclear staining are commonly associated with high-risk HPV infection, which may serve as useful surrogate biomarkers for identifying squamous cell carcinomas harboring HPV DNA.

Detecting the expression of human endogenous retrovirus E envelope transcripts in human prostate adenocarcinoma.

BACKGROUND: The expression of human endogenous retrovirus (HERV) mRNA and proteins was associated recently with diseases that include human malignancies. The authors report that, in the current study, transcripts encoding the envelope region of an HERV family, HERV-E, were expressed in human prostate carcinoma. METHODS: RNA was isolated from various prostate tissues and was tested for the expression of various HERV envelope (env) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, RNA in situ hybridization (ISH), and Northern blot analysis. Variants of HERV that appeared in prostate carcinoma tissues were sequenced, and HERV-E was expressed in prokaryotic and eukaryotic systems. RESULTS: In the current study, the authors found that the mRNA of the env gene of one particular family of HERVs, HERV-E, was expressed in some prostate carcinoma tissues (38.8% positive; n = 49 specimens) but not in normal prostate tissues using RT-PCR, RNA ISH, and Northern blot assays. The expression of HERV-E transcripts in prostate tumor epithelial cells was confirmed further by ISH using an HERV-E specific antisense probe. Approximately 50% of the cDNA of HERV-E obtained from prostate carcinoma specimens contained no stop codon and expressed proteins in prokaryotic or eukaryotic expression systems. Furthermore, the expression of both HERV-E and ERV3 (another class of HERV) was detected in the same prostate carcinoma tissues. CONCLUSIONS: The expression and distribution of multiple HERV-E endogenous retroviral elements in prostate carcinoma, but not in normal control specimens, suggests that they may serve as novel tumor markers for the early diagnosis and immunotherapy of patients with prostate carcinoma.

Quantitation of HERV-K env gene expression and splicing in human breast cancer.

Human endogenous retroviruses (HERVs) comprise up to 8% of the human genome. In previous studies, we demonstrated that type 1 HERV-K envelope (env) transcripts are expressed in most human breast cancers, but not in normal breast tissues. In the current study, we report that type 2 HERV-K env transcripts are also present in human breast cancers. By real-time RT-PCR, the expression of HERV-K env transcripts was 5-10-fold higher in breast cancer cell lines treated with estradiol and progesterone than in cells without treatment, and expression was significantly higher in most breast cancer tissues than in normal breast tissues. Furthermore, both types of HERV-K env transcripts were capable of being spliced into subgenomic env transcripts and various splice donor and acceptor sites were detected in breast cancers. The selective expression and distribution of multiple HERV-K endogenous retroviral element splice variants in breast cancer, but not in normal controls, suggests that they are novel breast tumor markers.

Endoscopic ultrasound guided fine-needle aspiration cytology of pancreatic carcinoma: a 3-year experience and review of the literature.

BACKGROUND: Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) of small pancreatic lesions that are undetectable by computed tomography has gained wide acceptance for the procurement of cells for diagnostic purposes. However, this technique is not without difficulty. The authors examined the sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of this technique in the evaluation of patients with pancreatic biliary duct strictures/masses. The authors were interested in reviewing their cases of pancreatic adenocarcinoma of ductal type and finding the sources of their false-negative cases. METHODS: A computer search was performed between January 1998 and July 2001. For the last 3 years, a total of 80 cases of suspected ductal adenocarcinoma of the pancreas was identified. Thirty-four patients (42%) underwent a subsequent Whipple procedure or biopsy. Cytologic and histologic correlation was performed in these cases. The rest of the 23 patients (29%) considered to be positive and the 23 patients (29%) considered to be negative underwent no subsequent biopsy and were followed clinically. Cases termed "suspicious" on cytology were considered positive and those termed "atypical cytology" were considered negative in the authors' final calculation. The causes of the false-negative diagnoses were evaluated carefully. RESULTS: Of the 34 cases followed with subsequent tissue biopsy or surgery; 12 were confirmed to be positive, 12 were confirmed to be negative, and 10 were considered to be false-negative. Previously identified cytomorphologic features of malignancy were used to review all cases. These features were: loss of the honeycomb pattern (100%), anisonucleosis (100%), nuclear contour irregularity (100%), a high nuclear/cytoplasmic ratio (100%), paranuclear chromatin clearing (77%), and the presence of prominent nucleoli in the absence of inflammatory cells (77%). The causes of the 10 false-negative cases were technical difficulty of procuring material in 6 cases, the nature of the lesion in 2 cases, and the scarcity of lesional tissue in 2 cases. CONCLUSIONS: Using strict cytoarchitectural and cytomorphologic criteria of malignancy for ductal pancreatic lesions previously described in the literature, the sensitivity of this technique at the study institution was 78% with a specificity of 100%. The PPV and NPV of this technique were 100% and 78%, respectively. The most common causes of the false-negative results in descending order were the technical aspect of the procedure, the size and nature of the lesion, and the scarcity of lesional tissue.

Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou tests using real-time polymerase chain reaction analysis.

BACKGROUND: The detection of specific human papillomavirus 16 (HPV-16) E6 and E7 oncogene transcripts may be a sensitive indicator of the direct involvement of viral oncogenes in the development of cervical neoplasia and carcinoma. The goal of this study was to determine the potential clinical uses of real-time polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) methods for evaluating HPV-16 E6 and E7 oncogene expression. METHODS: ThinPrep cervical samples were tested for expression of oncogenes of HPV-16 by real-time PCR or RT-PCR analysis and were compared with detection of expression by conventional PCR and RT-PCR analysis. Both sets of results were correlated with the cytologic diagnosis of the cervical samples. RESULTS: The presence of HPV-16 E6 and E7 DNA and RNA was observed only in HPV-16 positive cervical carcinoma cell lines but not in HPV-18 positive or HPV negative cell lines. The percentage positive for HPV-16 E6 or E7 DNA in a series of ThinPrep cervical cytologic samples (n = 348 samples) was 0% for negative samples (n = 45 samples), 9.7% for atypical squamous cells of undetermined significance (ASCUS; n = 144 samples), 16.9% for low-grade squamous intraepithelial lesion (LSIL; n = 118 samples), and 51.2% for high-grade intraepithelial lesion (HSIL; n = 41 samples). The copy numbers per nanogram for both DNA and RNA E6 and E7 were increased significantly as severity of the lesions progressed from ASCUS to HSIL, and RNA copy numbers were a more sensitive indicator of HPV-16 E6 and E7 expression than DNA copy numbers. The increase in copy numbers took place in a stepwise fashion from ASCUS, to LSIL, to HSIL. CONCLUSIONS: The detection of HPV-16 E6 and E7 expression by real-time RT-PCR or PCR analysis in ThinPrep cervical cytologic specimens may serve as a quick, reliable, and sensitive tool to identify a subset of patients who express HPV-16 oncoproteins.