Increased carcinoembryonic antigen expression in cervical intraepithelial neoplasia grade 3 and in cervical squamous cell carcinoma.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface protein that is overexpressed in a variety of human tumors and has been used as a tumor marker for disease progression in colorectal cancer patients. Recently, CEA has been used as a target for vaccine therapy against advanced CEA-expressing colonic adenocarcinomas. Previous reports have found elevated serum CEA levels in patients with cervical cancer, although this did not correlate with disease progression. In this study, cervical biopsy specimens from patients with normal histology, cervical intraepithelial neoplasia (CIN) grades 1 to 3, cervical squamous cell carcinoma (SCC), and adenocarcinoma were evaluated for CEA expression by immunohistochemistry by using the monoclonal antibody COL-1. Staining intensity was graded on a scale of 0 to 3 and was correlated with histologic diagnoses. CEA staining intensity was significantly increased in high-grade squamous lesions (CIN III and SCC) compared with normal cervical mucosa and CIN grades I or II (P <.0001). There was a linear correlation between grade of lesion and staining intensity (r = 0.71). CEA expression increased most significantly between CIN grades 2 to 3. Only 1 of 7 primary cervical adenocarcinomas expressed CEA. Only 1 of 10 patients with high CEA expression in their tumors by immunohistochemistry had elevated serum CEA. We thus have shown that lesional CEA expression increases in CIN 3 and SCC without elevations in serum CEA. CEA expression may be a useful diagnostic tool and a useful marker for identifying those at risk for progressive cervical neoplasia. HUM PATHOL 31:1357-1362.

Viral characteristics of human papillomavirus infection and antioxidant levels as risk factors for cervical dysplasia.

Genital human papillomavirus (HPV) infection is the major causal factor of cervical intraepithelial neoplasia (CIN). The potential role of nutrition as an additional, independent risk factor for CIN has not been appropriately addressed in the context of HPV. This case-control study evaluated the etiologic role of HPV in terms of viral type and load and examined the association between CIN and plasma levels of micronutrients adjusting for HPV. Cases (n = 378) with histo-pathologically confirmed CIN and controls (n = 366) with no history of abnormal Pap smears were recruited from colposcopy and gynecology clinics, respectively. Risk of CIN was significantly increased among women who were infected with multiple HPV types (odds ratio [OR] = 21.06), a high viral load (OR = 13.08) and HPV 16 (OR = 62.49). After adjusting for HPV positivity and demographic factors, there was an inverse correlation between plasma alpha-tocopherol and risk of CIN (OR = 0.15). Plasma ascorbic acid was protective at a high level of > or = 0.803 mg/dl (OR = 0.46). CIN was not associated with plasma retinol and beta-carotene levels. The effect of genital HPV infection on CIN development is highly influenced by oncogenic viral type and high viral load. Vitamins C and E may play an independent protective role in development of CIN that needs to be confirmed in prospective studies.

HPV 16 and cigarette smoking as risk factors for high-grade cervical intra-epithelial neoplasia.

Although genital human papillomavirus (HPV) infection is well established as the etiologic agent for cervical intraepithelial neoplasia (CIN), little is known about the cofactors involved in the development of high-grade lesions or the progression of low-grade to high-grade lesions. In our study of HPV-infected women with CIN (163 CIN I, 51 CIN II and 44 CIN III), women with CIN II or III were compared with those with CIN I for risk factors associated with high-grade lesions. After controlling for age, education, ethnicity and frequency of Pap smear screening, infection with HPV 16, but not high viral load or infection with multiple types, was associated with high-grade lesions (OR for CIN II = 11.96, OR for CIN III = 23.74). Risk of CIN III, but not CIN II, increased with number of cigarettes smoked per day (ORs = 1.49 and 3.35 for < or = 10 and > 10 cigarettes per day, respectively) and decreased with frequency of condom use during sex (ORs = 0.60 and 0.32 for women who used condoms occasionally/sometimes and most/all of the time, respectively). There were no associations between high-grade lesions and plasma levels of micronutrients (retinol, beta-carotene, alpha-tocopherol and reduced ascorbic acid). Our results indicate that infection with HPV 16 is associated with high-grade lesions. Additional cofactors, such as cigarette smoking, may be required as a carcinogen to advance HPV-infected cells toward neoplastic progression.

Stringent criteria for histological diagnosis of koilocytosis fail to eliminate overdiagnosis of human papillomavirus infection and cervical intraepithelial neoplasia grade 1.

Overdiagnosis of HPV infection in cervical biopsies results in increased health care costs and unnecessary surgical procedures. Stringent criteria for histological diagnosis of koilocytosis were evaluated, using molecular detection of HPV DNA (polymerase chain reaction and Southern blot hybridization) as gold standard. Colposcopic biopsy specimens from 511 patients were studied, including 76 with referral diagnoses of negative cervix and 241 with CIN 1 or koilocytosis. Referral diagnoses for low-grade lesions failed to distinguish between HPV-infected and uninfected patients. False-positive rate for prediction of HPV infection was 74.8%. Biopsy specimens reevaluated using stringent diagnostic criteria showed increasing prevalence of HPV infection among patients whose biopsy specimens showed negative (43.7%), minimal (52.4%), or definite (69.5%) features of koilocytosis (P = .001). Similarly, subjects infected with high viral load or oncogenic HPV infection were more likely to be identified (P = .004 and .04, respectively). Despite increased predictive value of stringent diagnostic criteria, significant number of patients diagnosed as having CIN 1/koilocytosis (34.0%) did not in fact have HPV infection. Because most low-grade lesions spontaneously regress, patients with histological diagnosis of CIN 1 or HPV infection should be observed for a period of several months before definitive ablative treatment is undertaken.

Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia.

BACKGROUND: Infection with human papillomavirus (HPV) type 16 (HPV16) is a major cause of high-grade cervical intraepithelial neoplasia (CIN). Experiments were planned to evaluate the role of cell-mediated immunity (e.g., lymphocyte proliferation) against HPV in the natural history of HPV-associated neoplasia and to identify antigenic sequences of the HPV16 proteins E6 and E7 against which an immune response may confer protection. METHODS: Forty-nine women with abnormal cervical cytology and biopsy-confirmed CIN were followed through one or more clinic visits. Lymphoproliferative responses of peripheral blood mononuclear cells to HPV16 E6 and E7 peptides were assessed in long-term (3-week) cultures. HPV DNA was detected in cervicovaginal lavage by means of polymerase chain reaction and Southern blotting. Disease status was determined by cervical cytologic examination and colposcopy. Reported P values are two-sided. RESULTS: Subjects with positive lymphoproliferative responses to E6 and/or E7 peptides were more likely to be HPV negative at the same clinic visit than were nonresponders (P = .039). Subjects who were negative for HPV and those with a low viral load were more likely to be responders than were those with a high viral load (P for trend = .037). Responses to N-terminal E6 peptide 369 were associated with absence of HPV infection at the same clinic visit (P = .015). Subjects with positive responses to E6 or E7 peptides at one clinic visit were 4.4 times more likely to be HPV negative at the next visit than were nonresponders (P = .142). Responses to E6 peptide 369 and/or E7 C-terminal peptide 109 were associated with an absence of HPV infection (P = .02 for both) and an absence of CIN (P = .04 and .02, respectively) at the next visit. CONCLUSIONS: Lymphoproliferative responses to specific HPV16 E6 and E7 peptides appear to be associated with the clearance of HPV infection and the regression of CIN.

Effects of beta-carotene and other factors on outcome of cervical dysplasia and human papillomavirus infection.

Women with histopathologically confirmed cervical intraepithelial neoplasia (CIN) were followed at 3-month intervals in a randomized double-blinded trial to evaluate the efficacy of beta-carotene to cause regression of CIN. Questionnaire data, plasma levels of micronutrients, and a cervicovaginal lavage for human papillomavirus (HPV) detection were obtained at each visit, and an endpoint biopsy was performed at 9 months. Sixty-nine subjects had a biopsy endpoint evaluation; 9 of 39 (23%) subjects in the beta-carotene group versus 14 of 30 (47%) in the placebo group had regression of CIN (P = 0.039). Independent risk factors for persistent CIN at 9 months included type-specific persistent HPV infection (OR = 11.38, P = 0.006) and continual HPV infection with a high viral load (OR = 14.25, P = 0.007) at baseline and 9 months, an initial diagnosis of > or =CIN II (OR = 6.74, P = 0.016), and older age (OR for > or =25 years = 4.10, P = 0.072). After controlling for these factors, the beta-carotene and placebo groups did not differ in risk for having CIN at 9 months (OR = 1.53, P = 0.550). Resolution of baseline HPV infection was significantly correlated with non-high-risk HPV types (RR = 2.94, P = 0.015), age <25 years (RR = 2.62, P = 0.014), and douching after sexual intercourse (RR = 3.02, P = 0.012), but not with randomization group. Our data indicate that a large proportion of mild CIN lesions regress; age and HPV infection play an important role in the natural course of CIN; and repeated HPV testing may have a value in distinguishing women who need aggressive treatment for CIN versus those who do not. Supplementation of beta-carotene does not appear to have a detectable benefit in treatment of CIN.

Morphologic pattern of tenascin as a diagnostic biomarker in colon cancer.

BACKGROUND: Immunohistochemical methods were used to study the pattern of expression of tenascin (TN) in invasive colon cancer and its relation to prognosis. METHODS: Sixty patients (29 males, 31 females) with a mean age of 77 years were studied. TN expression was evaluated by immunohistochemistry using paraffin-embedded tissue sections, TN expression levels were correlated with patient age, tumor stage, and survival. RESULTS: TN positivity varied from trace to 4+. Staining patterns were as follows: in well-differentiated cancer, TN fibers form thick bands around invading tumor glands. In poorly differentiated cancer, TN fibers had an interstitial pattern surrounding individual tumor cells. Using Cox's proportional hazard regression method, survival was significantly related to TN score (P < 0.0001) and stage of disease (P < 0.05). No significant relationship was found between survival and age (P = 0.375). CONCLUSION: Patients with more TN expression had better long-term survival than patients with no or weak TN expression. Pathologic and clinical entities in colon cancer have distinct immunohistochemical TN matrix patterns that may correlate with predictive value and long-term survival.

Induction of transforming growth factor beta-1 in cervical intraepithelial neoplasia in vivo after treatment with beta-carotene.

Transforming growth factor (TGF) beta1 is a potent growth inhibitor of epithelial cells. Loss of responsiveness to TGF-beta1 and/or loss of TGF-beta1 itself may be important in the progression of cervical intraepithelial neoplasia to invasive cervical cancer. Retinoids have antiproliferative effects on epithelial cells and have been used as chemopreventive and chemotherapeutic agents for several human cancers. There is evidence that retinoids exert their effects by promoting the induction of TGF-beta. The aim of this study was to determine whether the expression of TGF-beta1 was altered in patients enrolled in a clinical trial designed to test the therapeutic efficacy of beta-carotene, a carotenoid metabolized to retinol, in cervical intraepithelial neoplasia. Using an immunohistochemical technique, tissues were stained with two types of antisera that react with the intracellular and extracellular forms of TGF-beta1. Matched cervical biopsies taken from 10 patients before and after treatment with beta-carotene were immunostained simultaneously to allow direct comparison of relative staining intensity. A significant increase in intracellular TGF-beta1 immunoreactivity was noted in cervical epithelial cells in patients with cervical intraepithelial neoplasia after treatment with beta-carotene (P = 0.003). These results demonstrate regulation of a TGF-beta isoform in vivo in humans in response to beta-carotene administered as a chemopreventive agent.

Altered expression of transforming growth factor-beta 1 in cervical neoplasia as an early biomarker in carcinogenesis of the uterine cervix.

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cell growth, but can also stimulate stromal cell growth. Loss of responsiveness to TGF-beta 1 or loss of TGF-beta 1 itself may be important in the progression of cervical intraepithelial neoplasia (CIN) to invasive cervical carcinoma. METHODS. To examine the expression of TGF-beta in early stages of malignant transformation of the uterine cervix, paraffin embedded tissue samples from 11 patients with normal cervical epithelium, 15 with CIN I-III, 12 with microinvasive, and 18 with invasive squamous cell carcinoma were examined using an immunohistochemical technique. Tissues were immunostained with polyclonal antibodies that react with intracellular and extracellular forms of TGF-beta 1. RESULTS: Percent positive staining for the intracellular form of TGF-beta 1 was 100% for normal epithelium, 73.3% for CIN, and 44.1% for invasive carcinomas, (P = 0.002). Percent positive staining for the extracellular form of TGF-beta 1 was 63.6% for stroma underlying normal epithelium, 60% for stroma associated with CIN, and 94.1% for stroma surrounding invasive cancer (P = 0.007). CONCLUSIONS: Decreased expression of intracellular TGF-beta 1 in neoplastic epithelium and increased expression of extracellular TGF-beta 1 in stroma associated with invasive cervical carcinoma suggest that an early event in the neoplastic transformation of cervical epithelia] cells may involve the loss of TGF-beta 1. Tumor progression may be indirectly promoted by TGF-beta 1 secreted into or produced by supporting stromal elements.

Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia.

BACKGROUND: Cervical dysplasia, also referred to as squamous intraepithelial lesion (SIL) in cytology or cervical intraepithelial neoplasia in histopathology, is thought to have the potential to advance in progressive stages to cervical cancer. However, not all cases of SIL progress, and most of the mild lesions spontaneously regress. Factors that govern regression, persistence, and progression of SIL are poorly understood. PURPOSE: Our analysis sought to identify factors that determined persistence or regression of SIL. METHODS: Seventy subjects with histopathologically confirmed cervical dysplasia were followed at 3-month intervals for 15 months. At each visit, the cervix was evaluated by Pap smear and colposcopy, and exfoliated cervicovaginal cells were analyzed for human papillomavirus (HPV) DNA. For each subject, data from every two consecutive visits were grouped as a pair. Persistent SIL was considered present if a lesion was detected at a visit (t) as well as at the next visit (t + 1) and absent if a lesion was detected at visit t but not at visit t + 1. A statistical model for time-dependent data correlated persistent SIL with various risk factors. RESULTS: Age, ethnicity, education, sexual behavior, smoking, and the use of oral contraceptives did not correlate with persistent SIL. The risk of persistent SIL was associated with continual HPV infection in visits t and t + 1 (HPV positive by Southern blot analysis: odds ratio [OR] = 3.91, and 95% confidence interval [CI] = 1.58-9.65; HPV positive by polymerase chain reaction [PCR]: OR = 2.42, and 95% CI = 1.03-5.67) and a persistent high viral load (OR = 4.07, and 95% CI = 1.35-12.30). When typed by PCR, individuals with type-specific persistent infection in visits t and t + 1, and particularly those with a continual high viral load (OR = 4.97; 95% CI = 1.45-17.02), had the highest risk for persistent SIL compared with those with a low level of type-specific persistent infection or non-type-specific persistent infection. The presence of persistent HPV infection in visits t-1 (the preceding time interval) was also predictive of persistent SIL in visits t and t + 1, although the strength of association was weaker, suggesting that persistent HPV and SIL occur synchronously. CONCLUSION: HPV infection and its associated cervical lesions tend to occur concurrently, and type-specific persistent HPV infection, particularly with a high viral load, produces chronic cervical dysplasia. IMPLICATIONS: The natural history of genital HPV infection directly influences the prognosis of cervical dysplasia as measured by persistence of the lesion. Testing for HPV infection may be valuable in the clinical management of women with cervical dysplasia.

Genetic characterization of the human papillomavirus (HPV) 18 E2 gene in clinical specimens suggests the presence of a subtype with decreased oncogenic potential.

HPV 18 is associated with 2 divergent phenotypes: (i) aggressive cervical cancer and a preponderance of cancer relative to cervical intra-epithelial neoplasia (CIN) and (ii) benign warty lesions of the cervix. The E2 gene of HPV 18 encodes a regulatory protein that represses viral oncogene transcription and is involved in viral replication. Variation within the E2 gene of HPV 18 and its correlation with the morphologic grade of associated lesions were analyzed in a sample of 20 HPV 18-positive cervical specimens representing a spectrum of pathology from low-grade CIN to cervical cancer. An amplification HPV 18 E2 gene was present in 3 of 5 cancers, indicating that E2 disruption was not required for cancer development. Single-strand conformation polymorphism and PCR analyses revealed a high degree of polymorphism throughout the E2 gene. Direct DNA sequencing of both strands of a 154-bp fragment revealed a variability of 5.8%. Six intra-epithelial lesions contained alterations in common that account for 3.9% of the variation and appear to constitute a subtype. Within the 154-bp region, 2 of 3 cervical cancers and 0 of 12 intra-epithelial lesions were identical to the published HPV 18 sequence. DNA sequence analysis of a region extending into the E5 open reading frame revealed deletions in the E2/E5 intragenic region that were present in 50% of the members of the subtype. Our data demonstrate significant sequence variation within the E2 gene and suggest the presence of an HPV 18 subtype with decreased oncogenic potential.

Cell-mediated immune responses to E7 peptides of human papillomavirus (HPV) type 16 are dependent on the HPV type infecting the cervix whereas serological reactivity is not type-specific.

Forty-two women attending a colposcopy clinic for evaluation of abnormal cervical cytology and 13 normal controls were studied for the presence of lymphocyte proliferation (LP) cell-mediated immune (CMI) responses and serological reactivity to E7 peptides of human papillomavirus type 16 (HPV-16). HPV was typed by Southern blot hybridization of exfoliated cervicovaginal cell DNA. Positive LP responses (stimulation index > or = 5.0) to one or more E7 peptides were observed in 28.6% (12 of 42) of patients and 23.1% (three of 13) of controls. Of patients infected with HPV-16, -31 or -33, 63.6% (seven of 11) showed a positive LP response compared with 14.3% (two of 14) of women infected with other HPV types (P = 0.02), 17.6% (three of 17) negative for HPV (P = 0.02) and 23.1% (three of 13) of controls (HPV status unknown) (P = 0.05). C-terminal peptide 109 (amino acids 72 to 97) elicited positive LP responses in 45.4% (five of 11) of patients infected with HPV -16, -31 or -33 compared with 7.1% (one of 14) patients infected with other HPVs (P = 0.04), 5.9% (one of 17) of women negative for HPV (P = 0.02) and 7.7% (one of 13) of controls (P = 0.05). HPV-16 group-specific LP responses of borderline significance were also observed against E7 peptides 103, 105 and 108 (17-37, 37-54 and 62-80) (P = 0.07). ELISA reactivity (IgG) to E7 peptide 109 (72-97) was present in 7.7% (one of 13) of controls, 35.3% (six of 17) of HPV-negative patients, 42.9% (six of 14) of patients infected with other HPVs, and only 9.1% (one of 11) of patients infected with HPV-16, -31 or -33. CMI responses to C-terminal HPV-16 E7 peptide 109 (72-97) were thus significantly related to ongoing cervical infection with HPV-16 and closely related types, whereas serological reactivity to E7 peptides was not HPV type-specific.

Condyloma acuminatum with superficial spirochetosis simulating condyloma latum.

We report a case of a 12-year-old boy with a large perianal condyloma. The lesion was found to contain both spirochetes (Warthin-Starry stain) and human papillomavirus types 6 or 11 (in situ hybridization). Because of persistent negative serologies for syphilis and lack of other clinical stigmata of syphilis, the lesion was diagnosed as condyloma acuminatum with secondary involvement by spirochetes of probable intestinal origin. The case demonstrates the potential histologic similarities between condyloma latum and condyloma acuminatum, as well as the pitfalls of using the Warthin-Starry stain to establish the diagnosis of secondary syphilis.

Biologic characteristics of specific human papillomavirus types predicted from morphology of cervical lesions.

Human papillomavirus (HPV) DNA was detected by Southern blot hybridization in cervicovaginal lavage samples from 199 of 329 (60.5%) women attending a municipal hospital colposcopy clinic. Human papillomavirus was identified in 195 of 264 (73.9%) patients with a squamous intraepithelial lesion or cancer on biopsy or Papanicolaou smear (Bethesda system) compared with 11 of 65 (16.9%) without squamous intraepithelial lesion (P < .0001). The most common HPV type identified was HPV 16 (20.6% of positive samples), and 36.7% of isolates contained uncharacterized HPVs. Of women with cervical intraepithelial neoplasia (CIN) grade III or cancer, 23.4% were infected with HPV 16 compared with less than 4% with any other single HPV type. Based on biopsy diagnosis in patients infected with specific HPV types, HPVs 6 and 11 had low oncogenic potential; HPVs 18, 31, 35, and 45 had intermediate oncogenic potential; and HPVs 16 and 33 had high oncogenic potential. Hyperchromatic, unusually enlarged nuclei ("meganuclei"), and/or abnormal mitoses were found significantly more often in lesions infected with HPVs 16, 33, and 35 than in those infected with HPVs 6, 11, 18, 31, and 45, even in low-grade lesions, and may represent a histologic marker for HPVs with significant oncogenic potential. Human papillomavirus capsid protein was detected significantly less often by immunocytochemical staining in CIN I and CIN II lesions infected with HPVs 16 and 33 (8.3%) than in those infected with HPVs 6, 11, 18, and 31 (60%; P = .007), suggesting early abnormalities in cellular differentiation in lesions infected with highly oncogenic HPVs.

Polymerase chain reaction detection of human papillomavirus: quantitation may improve clinical utility.

A case-control study compared detection by polymerase chain reaction (PCR) specific for human papillomavirus (HPV) type 16 with restriction enzyme analysis and Southern blot hybridization detection of HPV type 16. Cervicovaginal lavage samples from 64 women with histopathologic evidence of a cervical squamous intraepithelial lesion and 55 samples from cytologically healthy women were studied. Several methods of PCR product analysis, including radioactive and nonradioactive probing, were compared. The sensitivity of HPV detection by PCR when the amplified DNA fragment was visualized on a gel was equivalent to those of detection by restriction enzyme and Southern blot analyses. Hybridization of the PCR product with radioactively or nonradioactively labeled oligonucleotide probes increased the sensitivity of HPV detection by 100-fold. However, an increase in the sensitivity of the assay preferentially identified low levels of the virus in cytologically healthy women. Therefore, the value of HPV detection in identifying women with cervical neoplastic disease was greater, and the odds ratio for the presence of a cervical squamous intraepithelial lesion was higher when the less sensitive modalities were used. These results suggest that quantitation of HPV by PCR may maximize the clinical significance of a positive test result. Further studies will be needed to determine the optimal level of virus detection which has the highest positive predictive value of clinical disease.