Human papillomavirus 16 DNA immortalizes two types of normal human epithelial cells of the uterine cervix.

Premalignant cervical lesions occur at the squamo-columnar junction and in endocervical epithelium and squamous ectocervical epithelium, in descending order of frequency. However, previously only ectocervical cells have been clearly shown to be immortalized in vitro by the oncogenic human papillomaviruses (HPVs). This report describes the immortalization of normal human ecto- and endocervical epithelial cells by the intact HPV 16 genome. Ectocervical epithelial cells (HEC) became immortalized (HEC-16) without crisis while endocervical cells (HEN) were immortalized (HEN-16) after undergoing crisis. HEN-16 and HEC-16 contained integrated HPV 16 DNA, expressed E6 and E7 mRNA, and were aneuploid and nontumorigenic. They also expressed cytokeratins in a pattern similar to their distinct normal parental cells. These results suggest that both squamous and simple epithelial cells of uterine cervix are targets for immortalization by HPV 16.     

The value of HPV DNA typing in the distinction between adenocarcinoma of endocervical and endometrial origin

AIMS: Distinguishing between adenocarcinomas of endocervical and endometrial origin histologically can be difficult, particularly in small biopsies. Most endocervical adenocarcinomas contain human papillomavirus (HPV) deoxyribonucleic acid (DNA) of 'high-risk' (HR) types, whereas this has not been consistently demonstrated in endometrial adenocarcinomas. The aim of this study was to determine whether HPV DNA testing could aid in this differential diagnosis. METHODS: The frequency of HPV DNA in paraffin-embedded tissue samples from 50 endocervical and 50 endometrial adenocarcinomas was investigated using polymerase chain reaction (PCR) amplification techniques involving (i) a screening HPV test followed by HPV DNA sequencing, and (ii) a test designed to detect HR genotypes 16, 18, 31, 33, 35, 45 and 58. Control specimens included cervical intraepithelial neoplasia (CIN) III lesions, squamous cell carcinomas (SCCs) of the cervix and lung, and colonic adenocarcinomas. Measures to minimise cross-contamination were implemented. RESULTS: The screening test followed by HPV DNA sequencing had the highest sensitivity. By this test HR HPV DNA was detected in 11 of 11 (100%) cervical intraepithelial neoplasia (CIN III) lesions, nine of 10 (90%) cervical SCCs, none of 10 (0%) colorectal adenocarcinomas and none of 10 (0%) SCCs of the lung. Thirty-nine (78%) endocervical adenocarcinomas contained HR HPV DNA, compared to one (2.0%) endometrial adenocarcinoma. CONCLUSIONS: The results suggest that HPV DNA testing could be a useful adjunct in distinguishing between endocervical and endometrial adenocarcinomas in curettings or small biopsy specimens.     

Cervical endometriosis: A potential pitfall in the evaluation of glandular cells in cervical smears

The use of the cytobrush and other endocervical sampling instruments has resulted in an increasing rate of detection and attention to glandular abnormalities of the cervix. Lesions that have been identified as look-alikes to endocervical gland dysplasia or adenocarcinoma in situ include squamous carcinoma in situ, atypical squamous metaplasia involving glands, and tubal metaplasia. In this report, we describe our recent experience with another condition that can mimic glandular abnormalities—cervical endometriosis. An in-depth review of the features seen in cervical smears from patients with cervical endometriosis is presented. Diagn. Cytopathol. 16:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Endocervical adenocarcinomas associated with lobular endocervical glandular hyperplasia: a report of four cases with histochemical and immunohistochemical analyses.

We report on four cases of endocervical adenocarcinoma associated with lobular endocervical glandular hyperplasia using histochemical and immunohistochemical analyses. The patients ranged in age from 59 to 67 years (mean 62 years). Chief complaints were watery vaginal discharge in two cases, genital bleeding in one and no subjective symptoms in one. Cytological examinations of the cervical smears revealed adenocarcinoma cells and benign-looking glandular cells with intracytoplasmic golden-yellow mucin in all cases. Radical hysterectomy was performed in three patients, and simple total hysterectomy was performed in one. From surgical specimens, three tumors were diagnosed as mucinous adenocarcinoma and one was adenocarcinoma in situ. All adenocarcinomas were located proximally on the cervix, and did not involve the transformation zone. Adjacent to carcinoma tissues in the cervix, lobular endocervical glandular hyperplasia was detected. The cells of lobular endocervical glandular hyperplasia were dominantly positive with neutral mucin, and immunohistochemistry revealed that these cells had prominent pyloric gland mucin (HIK1083). Focal immunopositivity for pyloric mucin was also observed in three adenocarcinomas. Either CEA or p53 were immunopositive in all adenocarcinomas and negative in the tissues of lobular endocervical glandular hyperplasia. Histopathological features of the present cases suggest that some endocervical adenocarcinomas may originate from lobular endocervical glandular hyperplasia.     

High concordance of results of testing for human papillomavirus in cervicovaginal samples collected by two methods, with comparison of a novel self-sampling device to a conventional endocervical brush

A user-friendly self-sampling method for collecting representative cervical cell material would lower the threshold for women to respond to the invitation for cervical screening. In the present article, we introduce such a device; we have evaluated its sensitivity and specificity to detect high-grade cervical intraepithelial neoplasia (CIN), via high-risk human papillomavirus (hrHPV) detection and liquid-based cytology (LBC), compared to endocervical brush samples obtained by gynecologists. Women who had a cervical smear reading of moderate dyskaryosis or worse or a repeat equivocal Pap smear result in the cervical screening program (n=64) and healthy volunteers (n=32) took a self-obtained sample at home prior to their visit to the gynecological outpatient department. At the outpatient department, an endocervical brush smear was taken, followed by colposcopy and biopsy whenever applicable. Both self-obtained samples and endocervical brush samples were immediately collected in Surepath preservation solution and used for LBC and hrHPV testing (by general primer-mediated GP5+/6+PCR). hrHPV test results showed a good concordance between the two sample types (87%; kappa=0.71), with sensitivities for prevalent high-grade CIN that did not differ significantly (92% and 95%; P=1.0). The hrHPV test on self-obtained samples proved to be at least as sensitive for high-grade CIN as cytology on endocervical brush samples (34/37 versus 31/37; P=0.5). LBC showed a poor concordance between self-obtained and endocervical brush samples (60%; kappa=0.27). In conclusion, self-obtained samples taken by this novel device are highly representative of the hrHPV status of the cervix. In combination with hrHPV testing, the use of this device may have implications for increasing the attendance rate for cervical screening programs.     

Immunocytochemical characterization of large granular lymphocytes in normal cervix and HPV associated disease.

A quantitative immunocytochemical study of large granular lymphocytes (LGLs) in the normal cervix and in human papillomavirus (HPV) associated disease was performed using a panel of monoclonal antibodies which included those for LGL surface markers CD56, CD16, and CD57. Only CD56-positive cells were found within the ectocervical epithelium and these cells increased in number in cervical intraepithelial neoplasia (CIN) in comparison with normal cervix. Examination of serial sections and double labelling suggests that these cells are CD3+, CD8+, CD56+, CD16+. The observed increase in number of this subset was not associated specifically with HPV infection but was related to CIN. Lymphocytes expressing all three LGL markers were found in the stroma and CD16(+)-positive cells clustered around endocervical glands with occasional cells extending into the endocervical epithelium. These results indicate that a small subset of LGLs which express T-cell markers is increased in number in CIN. Cells expressing classical NK markers are restricted to the stroma and are not found within the ectocervical epithelium.     

Adenoid basal carcinoma combined with invasive squamous cell carcinoma of uterine cervix: A case report of a 37‐year‐old woman and literature review

Adenoid basal carcinoma (ABC) is uncommon malignancy of the uterine cervix and it can be pure or combined with cervical intraepithelial lesions. There were less than 20 cases of ABC combined with invasive squamous carcinoma (mixed type) in English literature. These cases had similar properties as seen at postmenopausal women and diagnosed with abnormal cervical smear findings. Here we present a case of 37‐year‐old woman who suffered from spotting and received endocervical curettage. The pathological report revealed squamous cell carcinoma (SCC) of the cervix. The patient underwent type 3 radical hysterectomy and bilateral pelvic and para‐aortic lymph node dissection. The final pathological report revealed SCC coexisting with ABC. Human papillomavirus (HPV) 16,18 and others (11 types) were negative in both components of the mixed tumor by in situ hybridization detection. Our case was cytokeratin 7 negative, cytokeratin 8 positive and p63 positive which supports the hypothesis that mixed type cervical carcinoma originates from endocervical reserve cells.     

Surfactant protein D is present in the human female reproductive tract and inhibits Chlamydia trachomatis infection

Surfactant protein D (SP-D) is a lung collectin involved in innate host defence mechanisms in the lung. SP-D is also expressed at other mucosal sites throughout the human body. In the present study, we show that SP-D mRNA and protein are expressed in the human female reproductive tract. SP-D protein was localized in the apical portion of the reproductive epithelial cells. We also demonstrate that endometrial and endocervical cell lines and primary endocervical cells in culture produce SP-D mRNA and protein. Chlamydia trachomatis is an intracellular pathogen that infects the female reproductive tract, primarily the cervix, and is responsible for the most prevalent infectious disease in the USA. Untreated chlamydial infections of the female reproductive tract often result in sterility of the infected woman. Since SP-D protein is produced in cervical glands, we examined the effect of SP-D on chlamydial infection of cervical epithelial cells in vitro. We found that SP-D protein inhibits the infection of HeLa cells (an endocervical epithelial cell line) by C. trachomatis in a dose-dependent manner. We further demonstrate that the SP-D lectin-binding domain is involved in inhibiting infection of HeLa cells by Chlamydia. In conclusion, we detected SP-D in the female reproductive tract and determined that one of the functions of the SP-D protein may be to protect cervical epithelial cells from infection by C. trachomatis.     

Glandular lesions of the uterine cervix: the present state of our knowledge

The assessment of squamous lesions of the uterine cervix is a major preoccupation of diagnostic histopathologists and this can overshadow the recognition of significant abnormalities affecting the adjacent endocervical glands. This review gives an account of recent advances in our knowledge of the histology and histochemistry of endocervical glands. The review of benign lesions concentrates on those that continue to cause diagnostic difficulty. Adenocarcinoma in situ, lesser degrees of possibly pre-malignant cervical glandular atypia and histological subtypes of adenocarcinoma are outlined. The increasing awareness that glandular and squamous lesions often co-exist is emphasized. Finally, adenocarcinoma metastatic to the cervix is discussed.     

Occurrence of human papillomavirus 16 and 18 in smears from the two cervix regions of onco-gynecological patients in Slovakia

We evaluated the relevance of tests for Human papillomavirus 16 and 18 (HPV-16, HPV-18) in two cervix regions (exocervical and endocervical) separately. The total of 142 cervical smears obtained from 91 women in Slovakia attending onco-gynecological outpatient care were examined for the presence of HPVs by PCR with the general primers GP5 and GP6 (GP5/6). The HPV-positive smears were examined for the presence of HPV-16 and HPV-18 and the results compared with cytological assessment. In 73 HPV-positive smears, the number of cases with detected HPV-16 was about three times higher in exocervix and about two times higher in endocervix in comparison with number of cases with detected HPV-18. In the smears considered as normal by cytology, two times higher occurrence of HPV-18 in endocervical smears was found in comparison with exocervical ones. Eight patients were double-infected with HPV-16 and HPV-18, but no patient was infected with these HPVs in both cervical regions. This finding emphasized the importance of examination of both cervical regions separately. Overlooking of the endocervical canal for the close examination by cytology and PCR might increase the failure to detect HPVs associated with adenocarcinoma. Key words: Human papillomavirus; exocervix; endocervix; cytology; PCR.     

Application of immunohistochemistry in the evaluation of neoplastic epithelial lesions of the uterine cervix and endometrium

Routine use of immunohistochemistry has contributed greatly to the evaluation of neoplastic epithelial lesions of the uterine cervix and endometrium. This review highlights recognized diagnostic applications of these markers, addresses certain diagnostic pitfalls, and documents recent advances in the use of immunohistochemistry in the evaluation of cervical squamous intra-epithelial lesions (SILs), endocervical glandular neoplasia, basaloid cervical tumours, endometrial adenocarcinomas and their differential diagnoses. Increased MIB-1 staining is useful in distinguishing SILs and endocervical neoplasia (in situ or invasive) from benign mimics, such as cervical atrophy and tubal metaplasia, respectively. Immunohistochemisty with antibodies to vimentin, carcino-embryonic antigen (CEA) and oestrogen receptor (OR) may discriminate between invasive endocervical adenocarcinoma (ECA) and endometrial adenocarcinoma (EMC) in limited curettage specimens. ECAs tend to exhibit a vimentin-negative, CEA-positive, OR-negative profile, while the opposite is true for EMCs. Staining for MIB-1, OR and p53 may offer additional prognostic information in EMCs.     

Small Cell Undifferentiated Carcinoma of the Uterine Cervix: Histology, Ultrastructure, and Immunohistochemistry of Two Cases

Two clinical stage IB small cell undifferentiated carcinomas (SCUC) of the cervix were studied by light and electron microscopy and immunohistochemistry. Both cases occurred in women aged less than 31 years. Despite radical hysterectomy and external pelvic radiotherapy, both patients died of recurrent disease within 14 months after initial therapy. The tumors consisted of sheets of closely packed, uniform small, round to oval cells with hyperchromatic nuclei and scant indistinct cytoplasm. One case was associated with cervical squamous cell carcinoma. The neoplastic cells had few organelles and desmosome-like junctions and lacked mucinous or neurosecretory granules or tonofilaments. Immunohistochemistry failed to reveal S-100, CEA, neuropeptides or neuron-specific enolase.

SCUC probably arises either from basal cells of the cervical squamous epithelium, or gland cells of the endocervical epithelium, or still from subcolumnar endocervical reserve cells. Based on ultrastructure and immunohistochemistry, SCUC seems to represent the undifferentiated variant of small cell neuroendocrine tumors of the cervix.     

HPV-related cervical glandular lesions

The prevalence of premalignant and malignant endocervical glandular lesions is increasing in both relative and absolute terms. The majority of premalignant and malignant endocervical glandular lesions are aetiologically related to HPV infection. Neuroendocrine carcinomas of the cervix are also HPV related neoplasms that have an aggressive behaviour. This review concentrates on the latest classification and terminology of cervical glandular lesions and neuroendocrine carcinomas, their morphology and differential diagnosis and includes important practice points.     

Impact of patient characteristics on performance of nucleic acid amplification tests and DNA probe for detection of Chlamydia trachomatis in women with genital infections

The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women > or =20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women.     

The value of the Cytobrush for obtaining cells from the uterine cervix

A study was undertaken to evaluate the efficacy of the Cytobrush compared with the cotton-tipped applicator for obtaining cells from the uterine cervix. Four consecutive Papanicolaou smears were obtained in 1,000 patients during a single clinic visit. In every patient, a total of four smears on two slides were submitted: one slide contained endocervical material obtained with a cotton-tipped applicator and with the Cytobrush and the other slide contained ectocervical material obtained with both a cotton-tipped applicator and the Cytobrush. In endocervical smears, the Cytobrush technique produced numerous groups and clusters of cervical columnar cells in contrast to the use of the cotton swab. This was particularly true in older patients and pregnant women. In ectocervical smears, the detection of the squamous component was also higher in the Cytobrush smear than in cotton swab smears. However, the cell yield was not influenced by the cell sampling sequence in either endocervical or ectocervical specimens. It is the conclusion of this study that the utility of the Cytobrush technique for collecting specimens for endocervical and ectocervical smears is greater and provides a higher yield of cells than the cotton swab technique. Wider use of this simple procedure is encouraged.     

Expression of epithelial membrane and 3-fucosyl-N-acetyllactosamine antigens in cervix uteri with particular reference to adenocarcinoma in situ.

The staining patterns obtained with antiepithelial membrane antigen (anti-EMA) and the monoclonal antibody to 3-fucosyl-N-acetyllactosamine (AGF 4:48) in the uterine cervix in intraepithelial and invasive neoplasia were compared to determine a possible role in differential diagnosis of reactive and neoplastic conditions. Both early invasive and in situ adenocarcinoma stained equally intensely with both agents and both antibodies stained diffusely tubal metaplasia, endometrial lined glands, and even occasional areas of normal endocervical mucosa. It is concluded that these agents are unlikely to be of use in the routine histological differentiation of glandular and squamous cervical dysplasia or neoplasia, but immunostaining with anti-EMA may help differentiate between reactive and metaplastic changes in endocervical glands and adenocarcinoma in situ.     

LOW GRADE DIFFUSE FIBRILLARY ASTROCYTOMA OF THE CEREBRAL HEMISPHERES: LONG TERM FOLLOW UP AND p53 MUTATION ANALYSIS

Introduction and aims: Distinguishing between adenocarcinomas of endocervical and endometrial origin histologically can be difficult, particularly in curetting specimens with minimal material for examination. Endocervical adenocarcinomas have been shown to contain HPV DNA of certain 'high risk' subtypes, whereas this has not been consistently demonstrated in endometrial adenocarcinomas. The aims of this study were to look at whether HPV DNA typing could aid in this differential diagnosis.
Methods: The study investigated the frequency of HPV DNA in paraffin embedded tissue samples from endocervical and endometrial adenocarcinoma specimens using PCR amplification techniques designed to detect HPV DNA including high risk subtypes 16, 18, 31, 33, 45 and 58. Cases were selected from PathCentre and King Edward Memorial Hospital files, mainly curetting specimens with subsequent definitive hysterectomy. All cases were reviewed by a gynaecological pathologist. Control specimens included CIN III lesions, squamous cell carcinomas (SCC's) of the cervix and lung, and colonic adenocarcinomas. Measures to prevent cross contamination were implemented.
Results: HPV DNA was detected in 11 of 11 (100%) CIN III lesions, 9 of 10 (90%) cervical SCC's, 0 of 100 (0%) colorectal adenocarcinomas and 1 of 10 (10%) SCC's of the lung. 26 of 34 (76.5%) endocervical adenocarcinomas contain HPV DNA with 20 (55.6%) containing high risk subtypes, compared to 2 of 29 (6.9%) endometrial carcinomas, one with high risk subtype.
Conclusions: Preliminary results suggest HPV DNA typing could be a useful adjunct in distinguishing between endocervical and endometrial adenocarcinomas on curetting specimens, and possibly in the diagnosis of metastatic carcinomas of the cervix.     

Synchronous high-grade squamous intraepithelial lesion and adenocarcinoma in situ of cervix in a young woman presenting with hyperchromatic crowded groups in the cervical cytology specimen: report of a case

We report a 29-year-old woman who underwent routine gynecologic evaluation at a community clinic and had a cervical sample drawn for liquid-based cytologic evaluation. At cytology, many hyperchromatic crowded groups (HCG) were present, but a consensus could not be established whether the abnormal cells were primarily glandular or squamous with secondary endocervical glandular involvement. An interpretation of atypical endocervical cells, favor neoplastic, was rendered and biopsy advised if clinically appropriate. At biopsy, the cervix contained synchronous squamous cell carcinoma in situ, secondarily involving endocervical glands, and neighboring adenocarcinoma in situ. Immunohistochemistry for Ki-67 and p16(INK4A) crisply and precisely stained both the lesions, clearly separating them from the adjacent uninvolved mucosa. This case re-emphasizes the challenge associated with accurate evaluation of HCG at cytology, the significance of ancillary testing for surrogate markers of high-risk HPV (HR-HPV) infection, the need for adjunct testing for HPV-DNA in the setting of HCG at cervical cytology, and a recommendation to set up studies to evaluate the role of surrogate markers of HR-HPV infection in cytologic samples with HCG.     

Immunohistochemical localization of syndecan-1 in normal and pathological human uterine cervix

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.