Shaira Sahebali

P16INK4a as an adjunct marker in liquid‐based cervical cytology

Cytological screening for cervical cancer is hampered by high false negative rates. Inter‐observer reproducibility needs optimizing. The potential of p16INK4a as a biomarker for cervical lesions was examined in a study of liquid‐based cytology (LBC), HPV DNA testing by MY09/MY11 consensus PCR and type‐specific PCRs and p16INK4a immunocytochemistry on a series of 291 patients selected from routine screening. Comparison of the number of p16INK4a immunoreactive cells/1,000 cells exhibited a significantly higher mean count in HSIL (8.80 ± 1.13) than other cytological groups. The mean count of LSIL (1.09 ± 0.18) was significantly higher than that of the negative group (0.82 ± 0.40). ASC‐H and HSIL combined showed a significantly higher mean count (6.46 ± 1.17) than negative, ASC, ASC‐US and LSIL. The mean count of immunoreactive cells/1,000 cells was significantly higher in HPV16 positive samples (3.22 ± 0.72) than in samples containing infections with types of unknown malignant potential (0.83 ± 0.26) or HPV negative samples (1.17 ± 0.41). The mean count in infections with other high‐risk HPV types (2.55 ± 0.52) was significantly higher than that in HPV negative samples. Receiver‐operating characteristic curves yielded a test accuracy (area under curve) of 0.76, 0.79, 0.88 and 0.95 for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. Thresholds for 95% sensitivity were at 0.005, 0.007, 0.098 and 0.445 immunopositive cells/1,000 cells for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. The 95% specificity threshold for the detection of HSIL was at 1.87 immunopositive cells/1,000 cells. P16INK4a immunocytochemistry can be used as an adjunct to LBC in cervical screening, because it has a good diagnostic accuracy to discriminate HSIL and ASC‐H from other lesions. It could be used as a surrogate marker of high‐risk HPV infections.

Immunocytochemistry in liquid-based cervical cytology: analysis of clinical use following a cross-sectional study

Cytological screening for cervical cancer is hampered by imperfect sensitivity and low inter‐observer reproducibility. Human papillomavirus (HPV) testing lacks specificity as a primary screening method. Studies indicate that immunocytochemical detection of alterations caused by HPV in the host cells can optimise screening. Here, the potential of p16INK4a (cyclin‐dependent kinase inhibitor p16) and MIB‐1 (Ki‐67 proliferation marker) as adjunct molecular markers for cervical lesions was investigated in a prospective, cross‐sectional study of 500 samples in the framework of opportunistic screening in Flanders, Belgium. A consecutive series of 200 samples and 100 samples from the cytological categories ASC, LSIL and HSIL were investigated. Surepath samples were interpreted according to the Bethesda 2001 reporting system. HPV testing was done with MY09/MY11 consensus PCR. Immunocytochemistry for p16INK4a and MIB‐1 was performed with an automated staining protocol. The number of immunoreactive cells/1,000 cervical cells was assessed. There was a higher mean number of p16INK4A and MIB‐1 immunoreactive cells/1,000 cells in HSIL (4.06 ± 1.93 and 11.13 ± 2.83, respectively) compared to other cytological categories. Both markers showed a large spread in counts, for all categories. In cases of HSIL without immunoreactive cells for either marker, low cellularity and long‐term storage in water were often the cause of false negativity. This study confirms that positive staining for p16INK4a and MIB‐1 is highly correlated with presence of high‐grade lesions. These markers could be used as adjuncts to increase the sensitivity of cytological screening as well as the specificity of the HPV test. However, clear methodological standards are needed for optimal performance of immunocytochemistry in a clinical setting.

Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool?

Aims: To test the ability of Ki-67 to detect cytological lesions in a screening setting and its use as a surrogate marker of human papillomavirus (HPV) infection. Methods: A study of liquid based cytology, HPV DNA testing by MY09/MY11 consensus polymerase chain reaction (PCR), type specific PCRs, and Ki-67 immunocytochemistry on a randomly selected series of 147 patients. Results: Comparison of the number of Ki-67 immunoreactive cells/1000 cells in the different cytological groups showed that the HSIL group yielded a significantly higher mean count than did the other groups. The number of Ki-67 immunoreactive cells/1000 cells was significantly higher in HPV-16 positive samples than in samples containing infections with other high risk types. Receiver operating characteristic curves indicated a test accuracy (area under curve) of 0.68, 0.72, and 0.86 for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL), and high grade squamous intraepithelial lesions (HSIL), respectively. Thresholds for 95% sensitivity were 0.07, 0.08, and 0.15 Ki-67 immunopositive cells/1000 cells for ASCUS, LSIL and HSIL, respectively. The threshold for 95% specificity was 1.9 Ki-67 immunopositive cells/1000 cells. Conclusions: Ki-67 immunocytochemistry can be applied to liquid based cytology. The accuracy and diagnostic indices of the test are good when compared with those of other techniques. As part of a panel of screening procedures, it could be used as an adjunct to liquid based cytology to identify HSIL, and as a surrogate marker of HPV-16 infection.

Prior knowledge of HPV status improves detection of CIN2+ by cytology screening

OBJECTIVE The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions. STUDY DESIGN This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status. RESULTS Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status (P = .005) with limited loss of specificity (P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women (P < .001) vs older women (P = .729). CONCLUSION Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.

Role of Protein Biomarkers in the Detection of High-Grade Disease in Cervical Cancer Screening Programs

Since the Pap test was introduced in the 1940s, there has been an approximately 70% reduction in the incidence of squamous cell cervical cancers in many developed countries by the application of organized and opportunistic screening programs. The efficacy of the Pap test, however, is hampered by high interobserver variability and high false-negative and false-positive rates. The use of biomarkers has demonstrated the ability to overcome these issues, leading to improved positive predictive value of cervical screening results. In addition, the introduction of HPV primary screening programs will necessitate the use of a follow-up test with high specificity to triage the high number of HPV-positive tests. This paper will focus on protein biomarkers currently available for use in cervical cancer screening, which appear to improve the detection of women at greatest risk for developing cervical cancer, including Ki-67, p16INK4a, BD ProEx C, and Cytoactiv HPV L1.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears

Aims: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. Methods: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of β-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. Results: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. Conclusion: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

Effects of fixation on RNA integrity in a liquid-based cervical cytology setting

Aims: Despite many improvements, cervical cancer screening is still subject to shortcomings. Diagnostic accuracy may improve by using molecular biological techniques, requiring RNA of superior quality. This study determined the effect of SurePath fixation on RNA integrity to assess the suitability of clinical samples collected in this medium for RNA-based molecular assays. Methods: RNA isolation was performed on fresh and fixed HeLa cells and exfoliated cervical cells fixed in SurePath. The RNA integrity was evaluated by analysis of ribosomal RNA as an indicator of quality. The effect of SurePath preservation on PCR amplification was evaluated by real-time reverse transcriptase (RT)-PCR. Results: In contrast to unfixed cells, SurePath-fixed cells yielded less and severely degraded RNA, as shown by the absence of ribosomal RNA bands. RNA derived from SurePath-fixed cells showed poor real-time RT-PCR amplification characteristics, as evidenced by the absent correlation between threshold values and log cDNA concentration. Conclusions: Implementation of molecular biology in a clinical context is on the rise and may alleviate shortcomings in current screening and diagnostics. This study shows that SurePath fixation gives rise to highly fragmented RNA with insufficient quality for further reliable analysis by standard real-time RT-PCR applications. The increasing prominence of molecular screening stresses the importance of this finding, which must be considered in relation to choice of an appropriate liquid-based cytology system.

An unusual case of ectopic tungiasis with pseudoepitheliomatous hyperplasia

Tungiasis is caused by the penetration of the female sand flea Tunga penetrans into the epidermis, and subsequent hypertrophy of the parasite. In most cases lesions are confined to the feet. During a cross-sectional study, an unusual case of ectopic tungiasis in the inguinal area was detected. Histological examination of tissue samples showed a remarkable pseudoepitheliomatous aspect of the epidermis. Clinical features and differential diagnoses are discussed.

Cervical cytological screening and human papillomavirus DNA testing in Flanders.

Abstract The causal relationship between genital human papillomavirus (HPV) infection and cervical dysplasia/ carcinoma has been recognised for some time. The aim of this study was to document the occurrence and distribution of HPV infection in the five provinces of the Flemish region in Belgium and to correlate the HPV DNA test results with the cytological results on simultaneously performed thin layer preparations of cervical cells. Out of a total screened group of 105107 samples, 1978 samples with cytological abnormalities were tested for HPV DNA using the MY09/MY11 consensus PCR. The mean age of the whole group was 36.9 years. The LSIL group, with a mean age of 33.6 years, was significantly younger than the other groups. There was no significant difference in HPV prevalence among the provinces. In four out of five provinces the HPV prevalence reached 100% in high-grade lesions. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology (17.9% WNL < 51.1% ASCUS < 83.8 % LSIL < 97.2% HSIL). Three peaks of HPV DNA positivity were observed, a first at 22 yrs (82%), a second at 47 yrs (60%) and a third in women older than 65 yrs (52%). These results shed more light on HPV prevalence in Flanders and show that the MY09/MY11 consensus primer based detection system is very suitable for the detection of HPV infections in Flanders.

Stromal issues in cervical cancer: a review of the role and function of basement membrane, stroma, immune response and angiogenesis in cervical cancer development

The carcinogenesis of cervical carcinoma implies an intricate interplay of neoplastic, human papillomavirus infected epithelial cells and stromal tissue, in which different factors have distinct but interacting influence. Persistent infection with an oncogenic human papillomavirus type may lead to epithelial dysplasia with progressive severity. To access the adjacent stromal tissue, tumour cells have to breach the basement membrane. The stroma partly controls tumour growth, invasion and angiogenesis. Last but not least there is considerable influence of the immune response. In this review we describe the importance of various stromal factors in carcinogenesis of cervical cancer.