Seroresponses to human papillomavirus types 16, 18, 31, 33, and 45 virus-like particles in South African women with cervical cancer and cervical intraepithelial neoplasia

The aim of the study was to determine the prevalence of antibodies to human papillomavirus (HPV) types 16, 18, 31, 33, and 45 in woman in Cape Town with cervical intraepithelial neoplasia (CIN) (n = 95), cervical cancer (n = 40), female blood donors (n = 95) and children (n = 110). The enzyme-linked immunosorbent assay (ELISA) made use of baculovirus synthesised HPV virus like particles (VLPs) as antigen. Antibodies to at least one HPV type were detected in sera from 75% of cancer patients, 71.6% of CIN patients, 44.2% of blood donors and 27.3% of children. Sera from 95 women with CIN were compared with age-matched female blood donors. There was a significant association of seropositivity to VLP-16 (P = 0.006) and VLP-45 (P = 0.008) with CIN compared with the blood donors. There was also a significant difference in the seropositivity of women with CIN to any of the five virus-like particle (VLP) types compared to the blood donors (P = 0.0002: OR = 3.2). Thirty-nine of sixty-nine (56.5%) women with CIN were found to be HPV-16 DNA positive. The average age of women in this group that were VLP-16 seropositive was 34 years and those found to be VLP-16 seronegative was 52 years of age. Antibodies to all five VLP types were detected in these populations, thus an ideal vaccine should induce protection from infection by a wide range of HPV types.     

Comparative evaluation of nm23 and p16 expression as biomarkers of high-risk human papillomavirus infection and cervical intraepithelial neoplasia 2(+) lesions of the uterine cervix

Benevolo M, Terrenato I, Mottolese M, Marandino F, Muti P, Carosi M, Rollo F, Ronchetti L, Mariani L, Vocaturo G & Vocaturo A
(2010) Histopathology 57 , 580–586
Comparative evaluation of nm23 and p16 expression as biomarkers of high‐risk human papillomavirus infection and cervical intraepithelial neoplasia 2⁺ lesions of the uterine cervix Aims: To investigate the clinical role of nm23 expression in identifying both high‐risk human papillomavirus (HR‐HPV) and high‐grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2⁺ (CIN2⁺)], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR‐HPV infected cervical lesions. Methods and results: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low‐ and high‐grade lesions and squamous carcinomas (SC). HR‐HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico‐vaginal samples of the same patients. In detecting CIN2⁺, p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P < 0.0001). Concerning HR‐HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% versus 47%, P <0.0001), although the sensitivities were comparable (71% versus 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. In contrast, we observed a significant association between p16 overexpression and HPV16/18 genotypes. Conclusions: We confirm the diagnostic value of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV‐E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2⁺ or HR‐HPV infection.     

Large cell neuroendocrine carcinoma of the uterine cervix: a report of a case with coexisting cervical intraepithelial neoplasia and human papillomavirus 16.

Large cell neuroendocrine carcinomas (LCNECs), one of the four newly categorised endocrine tumors of the uterine cervix, are unusual and aggressive tumors. The present report describes a case of LCNEC diagnosed at an early stage and associated with cervical intraepithelial neoplasia (CIN). The LCNEC showed organoid and trabecular growth patterns and was positive for chromogranin and synaptophysin. The CIN lesion was of a high grade and was negative for these neuroendocrine markers. Polymerase chain reaction (PCR) using genomic DNA extracted from archival tissue demonstrated human papillomavirus (HPV) type 16 DNA in both the LCNEC and CIN lesions. These histological, immunohistochemical and PCR findings suggested that the LCNEC lesion was distinct from the CIN lesion and that both resulted from the carcinogenic field effect of HPV 16.     

Prognostic value of human papillomavirus types 16 and 18 DNA physical status in cervical intraepithelial neoplasia

The aim of this work was to assess the value of the physical status of human papillomavirus (HPV) DNA as a disease marker for cervical cancer development in a set of 248 DNA samples previously genotyped as HPV 16 or 18, by calculating the E2/E6 ratio through real-time PCR. There was a significant difference in integration status according to disease grade for both genotypes (p <0.001). Furthermore, especially for HPV 18, determining the DNA physical status could be a useful biomarker in predicting cervical cancer risk development, with a lower E2/E6 ratio clinically associated with the development of a precancerous lesion.     

Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix

Many reports have shown a link between human papillomavirus (HPV) and cervical squamous neoplasia. However, the association of HPV with cervical adenocarcinoma has been studied less extensively. The authors evaluated the presence of HPV-DNA in 106 patients with adenocarcinoma of the uterine cervix by in situ hybridization, using 35S-labeled probes for HPV 16 DNA and HPV 18 DNA. The overall prevalence of HPV-DNA was 18% (19 of 106). HPV 16 was present in 2 (2%) cases, HPV 18 was observed in 15 (14%) cases, and both HPV 16 and HPV 18 were found in 2 (2%) cases. There was a correlation between HPV-DNA positivity and tumor stage (P less than 0.01) and tumor size (P less than 0.05), but there was no relationship between HPV-DNA positivity and tumor differentiation, proliferation (S-phase fraction), ploidy, lymph node metastases, or five-year survival rate. These results suggest that HPV 18 DNA is associated with cervical adenocarcinoma but the presence of HPV 18 has no influence on overall survival.     

Neutralizing antibodies against human papillomavirus types 16, 18, 31, 52, and 58 in serum samples from women in Japan with low-grade cervical intraepithelial neoplasia

We have very limited information on serum neutralizing antibody in women naturally infected with the human papillomaviruses (HPVs) that are causally associated with cervical cancer. In this study, serum samples collected from 217 Japanese women with low-grade cervical intraepithelial neoplasia were examined for their neutralizing activities against HPV16, -18, -31, -52, and -58 pseudovirions. Eighty-four patients (39%), 35 patients (16%), 17 patients (8%), and 1 patient were positive for neutralizing antibodies against one, two, three, and four of these types, respectively. Presence of neutralizing antibody did not always correlate with detection of HPV DNA in cervical swabs collected at the time of blood collection. The neutralizing titers of the majority of sera, ranging between 40 and 640, were found to be conserved in the second sera, collected 24 months later, independently of emergence of HPV DNA in the second cervical swabs. The data strongly suggest that HPV infection induces anti-HPV neutralizing antibody at low levels, which are maintained for a long period of time.     

Tenascin in human papillomavirus associated lesions of the uterine cervix.

The immunohistochemical expression of tenascin was studied in 80 morphologically diagnosed condylomas and cervical intraepithelial neoplasia (CIN) lesions. The results were compared with the human papillomavirus (HPV) DNA subtype, which was determined by HPV dot blot and in situ hybridisation. Tenascin mRNA synthesis was also determined in 10 selected cases by in situ hybridisation. No statistically significant association was found between tenascin expression and the degree of dysplasia or the HPV subtype. There was, however, a strong correlation between the extent of tenascin immunoreactivity and the degree of inflammation. Synthesis of tenascin mRNA was detected in basal keratinocytes and in fibroblasts by in situ hybridisation. The lack of association between the grade of CIN and tenascin expression precludes its use as a marker of premalignancy in CIN.     

Prevalence of human papillomavirus types 6, 11, 16, 18, 31, and 33 in a cohort of Greek women

To study HPV prevalence and HPV types 6, 11, 16, 18, 31, and 33 distribution in cervical smears in a cohort of Greek women. One thousand six hundred thirty-six samples were cytologically evaluated and molecularly analyzed, by PCR based assay. Abnormal cytology was identified in 997 women and 75.4% of them were HPV DNA positive, while 639 had normal cytology and 24.6% were HPV DNA positive. HPV was detected in 62.9% of 256 ASCUS smears, 89.3% of 516 LSIL, 86.7% of 60 HSIL and 47.3% of 165 with cervical carcinoma. Overall, HPV 11 was the most common type (13.4%), followed by 18 (10.3%), 6 (7.2%), 16 (6.4%), 31 (3.4%) and 33 (3.4%). Multiple infections with two (11.3%) or more types, primarily 11 and 18 (4.8%), were also identified. Low-risk types 11 and 6 were common in ASCUS (36.6% and 26.4%, respectively), and high-risk types 16 and 18 in HSIL (42.3% and 30.8%, respectively) and in cancer (51.3% and 41%, respectively). Multiple infections were detected in 2.2% of normal and 31.7% of HSIL. HPV prevalence was 75.4% in abnormal and 24.6% in normal cervical smears. HPV 16 and 18 were the most common types in cancer. Single infection with type 11 and multiple infections with 11 and 18 were more frequent.     

Tenascin expression in intraepithelial neoplasia and invasive carcinoma of the uterine cervix

OBJECTIVE: To determine whether the expression of the matrix protein tenascin (TN) is of diagnostic or prognostic value in cervical intraepithelial neoplasia (CIN). DESIGN: Tenascin expression was evaluated in 75 formalin-fixed, paraffin-embedded biopsy and surgical specimens of the uterine cervix. Specimens included 15 low-grade squamous neoplastic lesions (CIN I), 30 high-grade squamous neoplastic lesions (CIN II and CIN III), 5 microinvasive carcinomas, and 15 invasive squamous carcinomas. Five normal cervices and 5 examples of cervicitis were used as controls. Expression of TN was studied by immunohistochemistry with a monoclonal mouse anti-human tenascin antibody. Tenascin expression in the basement membrane and in the stroma was arbitrarily graded as normal or slightly, moderately, or markedly increased. RESULTS: In the normal cervix, TN formed a thin band along the basement membrane of the squamous epithelium, except for the transformation zone, where the bands splintered and delicate TN fibers were present in the adjacent stroma. In cervicitis, TN bands were splintered in the basement membrane and the protein was weakly expressed in the stroma infiltrated by inflammatory cells. In the 45 CIN lesions, regardless of grade, the TN bands in the basement membrane were slightly (25 cases) or moderately (20 cases) increased. In CIN lesions with chronic stromal inflammation, a slight increase in stromal staining was observed, similar to the findings in cervicitis. In microinvasive and frankly invasive squamous cell carcinomas, TN expression was markedly increased in the basement membrane and in the stroma surrounding the invasive nests of cancer cells. CONCLUSION: Tenascin expression may be of value in the assessment of early stromal invasion in cancer of the uterine cervix. Tenascin expression is of no value in distinguishing various grades of CIN and, therefore, is not a predictor of future behavior.     

Polymorphism of the capsid L1 gene of human papillomavirus types 31, 33, and 35

The L1 gene encodes for the major capsid protein of human papillomaviruses (HPV). There is limited information on the polymorphism of L1 for types related to HPV‐16. This report explores the polymorphism of L1 in phylogenetically related types 31, 33, and 35 compared to HPV‐16. Genital specimens collected from 732 HIV‐seropositive and 323 HIV‐seronegative women were screened for HPV DNA with consensus L1 PCR. Cervical samples positive for HPV‐16 (n = 74), HPV‐31 (n = 78), HPV‐33 (n = 37), and HPV‐35 (n = 58) were further characterized by PCR‐sequencing of the complete L1 gene. The number of nucleotide substitutions within L1 ranged from 19 for HPV‐33 to 52 for HPV‐31. The ratio of the number of variants/number of isolates tested was higher for HPV‐31 (56.4%, P = 0.05) and HPV‐35 (60.3%, P = 0.04) compared to HPV‐16 (40.5%), while this ratio was lower for HPV‐33 (24.3%), although not significantly (P = 0.14). The maximal distance between HPV variants was greater in the five putative surface‐exposed loops of L1 than in sequences outside the loops (P < 0.01). Synonymous variations were encountered in 1.7% (95% CI 1.1–2.3) of nucleotides inside the L1 loops and 2.4% (95% CI1.2–3.7) of nucleotides outside the L1 loops. Non‐synonymous variations were encountered in 1.8% (95% CI 1.1–2.5) of nucleotides within the L1 loops and 0.2% (95% CI 0–0.4) of nucleotides outside the loops. dN/dS ratios were below 1.0 in extra‐loop and intra‐loop regions, but they were lower in extra‐loop regions. These results suggest that sequences within and outside the hypervariable loops of L1 were under selective constraint. J. Med. Virol. 82: 1168–1178, 2010. © 2010 Wiley‐Liss, Inc.     

Seroresponses to virus-like particles of human papillomavirus types 16, 18, 31, 33, and 45 in San people of Southern Africa

Virus-like particles (VLPs) of the high-risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 were used as antigen in enzyme-linked immunosorbent assay (ELISA) to determine the prevalence of serum IgG in a group of San people originally from Namibia, now residing in South Africa. The San children had low seroprevalence to all VLP types, but 26/115 (22.6%) of the children were seropositive to at least 1 VLP type. Among the adults, seroprevalence was significantly higher. The seroprevalence of antibodies in 101 San women to VLP-16 was 16.8%, VLP-18 18.8%, VLP-31 12.9%, VLP-33 17.8%, and VLP-45 22.8%. Five of the 11 men were seropositive: 2 for VLP-31, 1 for VLP-18, 1 for VLP-33, and 1 for VLP-45. Seroreactivity appeared to be type specific, except possibly to VLP-18 and -45. Of the adults, 50.5% were seropositive to at least 1 VLP type and 24.8% were seropositive to >1 VLP type. From this study, it is concluded that the San people are exposed to HPV-16, -18, -31, -33, and -45, with antibodies to VLP-45 being the most prevalent.     

Comparison of Southern transfer hybridization and dot filter hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35

A commercial dot filter hybridization kit (Virapap Kit) was compared with Southern transfer hybridization for the detection of seven types of human papillomavirus (HPV) in cervical specimens from 450 consecutive females attending a sexually transmitted diseases clinic. In comparison with Southern transfer hybridization, performed with the same probes used in the dot filter kit, the sensitivity, specificity, and positive and negative predictive values of dot filter hybridization were 90%, 94%, 74%, and 98%, respectively. Among patients with cervical cytologic dysplasia, HPV DNA was detected in 44% by dot filter hybridization and in 35% by Southern transfer hybridization. Although 26% of specimens positive by dot filter hybridization were not confirmed by Southern transfer hybridization, cervical dysplasia was detected in 5 (25%) of 20 with HPV DNA detected by dot filter hybridization alone, compared with 25 (8%) of those with no definitive evidence of HPV by either method (P = 0.009) and with 16 (30%) of 53 with HPV DNA detected by both methods (P = 0.7). The kappa statistic for interobserver and intraobserver reproducibility for interpretation of blots was similar for the two methods. The dot filter hybridization method evaluated appears to be a satisfactory alternative to Southern transfer hybridization for detection of HPV DNA.     

Characterization of gut-derived intraepithelial lymphocyte (IEL) residing in human papillomavirus (HPV)-infected intraepithelial neoplastic lesions

Citation Kojima S, Kawana K, Fujii T, Yokoyama T, Miura S, Tomio K, Tomio A, Yamashita A, Adachi K, Sato H, Nagamatsu T, Schust DJ, Kozuma S, Taketani Y. Characterization of gut‐derived intraepithelial lymphocyte (IEL) residing in human papillomavirus (HPV)‐infected intraepithelial neoplastic lesions.Am J Reprod Immunol 2011; 66: 435–443 Problem Mucosal T cells are the most likely direct effectors in host anti‐human papillomavirus adaptive immunity and regression of cervical intraepithelial neoplasia (CIN) lesions. There are no studies addressing intraepithelial lymphocytes (IELs) in CIN lesions. Method of study Cervical lymphocytes were collected using cytobrushes from patients with CIN and analyzed by FACS analysis. Comparisons were made between populations of cervical T cells in CIN regressors and non‐regressors. Results A median of 74% of cervical lymphocytes were CD3⁺ T cells. Populations of integrin αEβ7⁺ IEL in CIN lesions varied markedly among patients (6–57%). Approximately half of integrin β7⁺ T cells were CD45RA‐negative memory T cells. The number of integrin αEβ7⁺ cells among cervical T cells was significantly higher in CIN regressors when compared to non‐regressors. Conclusion Higher cervical IEL numbers are associated with spontaneous regression of CIN. Accumulation of cervical integrin αEβ7⁺ IEL may be necessary for local adaptive effector functions.     

The p16INK4a protein: a cytological marker for detecting high grade intraepithelial neoplasia of the uterine cervix

The identification of a small percentage of high grade cervical intraepithelial neoplasia (HGCIN) among patients with a diagnosis of atypical squamous cells of undetermined significance (ASC-US) and low grade squamous intraepithelial lesion (LSIL) is one of the difficulties in cytology based cervical cancer screening. p16INK4a is a surrogate marker for the initiation of HPV mediated cervical carcinogenesis. This article describes the detection of the protein p16INK4a by immunocytochemistry coupled with the use of a nuclear score to differentiate abnormal basal cells from metaplastic or atrophic cells. The results of a pilot series of 210 liquid based cytology (LBC) specimens of which 108 were considered normal, 52 with a diagnosis of LSIL and 50 with a diagnosis of high grade SIL are described. The second series includes 137 LBC specimens with an ASC-US diagnosis and 88 with a LSIL diagnosis with an histological correlation. The overall sensitivity for the diagnosis of HGCIN using for the labeled squamous cells a nuclear score superior to 2 was 96% and the specificity was 83%. The sensitivity in the ASC-US was 95% and the specificity was 84%, in the LSIL group 100% and 81%, respectively. These data suggest to study a large series of LBC smears with a diagnosis of ASC-US and LSIL to confirm its efficacy of predicting the presence of an HGCIN.