Immunohistochemistry
Introduction
The Human Papilloma virus (HPV) is an extremely common group of viruses that are found worldwide. There are more than 100 types of HPV worldwide. 13 types of HPV are associated with cancer. They are also termed as high risk type. (WHO, 2013). HPV is transmitted through sexual contact. More than 90% of cases are infected shortly after sexual activity. Cervical cancer is associated with HPV infection that are acquired through sexual contact. 70% of cervical cancer cases are caused by two types of HPV. HPV 16 and HPV 18 are the two strains associated with cervical cancer. (WHO, 2013). HPV is also associated with cancers of the vagina, penis, anus, anus, and vulva. There are around 530,000 new cases of cervical cancer every year. (WHO, 2013). Cervical cancer is the second leading most common cancer found in women. (WHO, 2013). More than 270,000 women die of cervical cancer every year. Most of the deaths occur in low and middle-income countries.
Immunohistochemical method to identify a strain of human papilloma virus (HPV) associated with cervical cancer from a colposcopy sample from a patient
Cervical cancer screening for pre-cancer and cancer in women is highly recommended. WHO recommends cancer screening for every women aged 30 to 49 at least once in a lifetime. There are three different types of screening tests that are currently used in major diagnostic labs and hospitals. Liquid-based cytology (LBC) and conventional (Pap) test, visual inspection with Acetic Acid (VIA), and HPV testing for high-risk HPV types. The paper highlights the different screening techniques (Immunihistochemical) to identify the strain of HPV associated with cervical cancer. (Soma M, & Kamaraj S. 2010)
The Papanicolaou-stained (Pap) smear is considered as the primary method for detection of high-risk HPV. The method was developed by pathologist George Papanicolaou. The method was developed before the cause of cervical cancer was known. The reporting classification of Pap smear has changed over time. The Bethesda System is currently used as the reporting system. (Table 1). The system was introduced in 1989, and updated in 1999. The Bethesda system was introduced to replace the cervical intraepithelial neoplasia (CIN) system. The Bethesda system was developed to understand the advanced cervical neoplasia. The system also introduced descriptive diagnostic histologic terminology. (Soma M, & Kamaraj S. 2010). According to the modified (2001 version) of the Bethesda system, there are four categories of squamous cell abnormalities in cervical cancer. ASC (atypical squamous cells), HSIL (high-grade squamous intraepithelial lesions), LSIL (low-grade squamous intraepithelial lesions), and squamous cell carcinoma are the four types of cell abnormalities found in cervical cancer. (Soma M, & Kamaraj S. 2010). There was confusion in the ASC category. The updated version of ASC contains two subcategories, atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells, cannot exclude HSIL (ASC-H). The Pap smear procedure is known for its strength and limitations. The rate of false-negatives in Pap smear tests are high (20% to 30%). (Soma M, & Kamaraj S. 2010). Clumping of cells increase the chances of false-negative results. Human error is considered as the biggest threat to accurate perception in Pap smear readings. The average Pap smear slides contain 50,000 to 250,000 cells that are examined by trained professionals. However, the risk of human error in such cases are high. The Clinical Laboratory Improvement Act (CLIA) established national guidelines to restrict the number of slides read by technicians to not more than 100 per day.
Monolayer Cytology
Many methods have been developed to collect and process Pap smear specimens. Most of the methods are known to reduce the false-negatives observed in conventional Pap smear cytology. The new method involves includes the collection of samples in a preservative solution. Cellular structure in such advanced methods are preserved since the cells are immediately fixed. The new method also involves the use of a cervical brush that helps in gathering twice the amount of epithelial cells. The advanced method also involves the preparation of slides under the control of the cytology laboratory. Uneven layering is avoided in the new method. The uniform monolayer used in the new method helps technicians to read slides easily. Interpretation of results is easy in the case of monolayer cytology. The process also prevents drying of cells on the slide. The methods also removes contaminants like fungus, bacteria, mucus, and red blood cells. There are currently two FDA approved methods of monolayer cytology methods. The PrepStain system (formerly the AutoCyte PREP system) and the ThinPrep Pap Smear methods are approved by the FDA. (Soma M, & Kamaraj S. 2010). The methods used in the preparation of slides in both the systems are different but the underlying principles are the same. Ethanol based preservative solution is used in the PrepStain system. Density gradient centrifugation is utilized to enrich the sample and remove non-diagnostic and inflammatory debris. The sample is placed onto an adhesive-coated microscopic slide. The method involves automated techniques to stain each slide with a separate Papanicolaou stain. The method is known to produce highly accurate and precise results. On the other hand, the ThinPrep Pap Smear method involves a buffered alcohol preservative solution. The preserved sample is gently dispersed by high-speed rotation. Vacuum is utilized to draw the suspension through a polycarbonate filter. A microprocessor ensures that equal number of cells are deposited on the filter. The next involves the transfer of filtered cells onto a 20mm monolayer microscopic slide followed by Papanicolaou staining. (Soma M, & Kamaraj S. 2010).
Monolayer cytology is known for its high cost and accuracy. However, more than 50 peer-review articles recommend the use of monolayer cytology method to detect pancreatic lesions and HPV strains associated with cervical cancer. (Soma M, & Kamaraj S. 2010). Most of the peer-reviewed articles demonstrated high accuracy and precision of monolayer cytology over conventional Pap smear methods. Moreover, monolayer cytology method increased the detection of epithelial abnormalities from 4% to 115% in infected patient populations. (Lee, K. R., et al; 1997). On comparing conventional Pap smear methods and monolayer cytology methods, monolayer cytology had high accuracy in detecting the presence of dysplasia in colposcopically directed biopsies. (Sheets, E. E., et al; 1995). Computer-assisted systems have been utilized to evaluate Pap smears. The FDA had approved two device, the PapNet and AutoPap 300QC to ensure objective and consistent evaluation of Pap smears with minimal human effort. Researchers also utilize advance methods to improve Pap smear diagnosis. The BenchMark performs immunohistochemistry stains on ThinPrep Pap Smear cytology samples. A probe present in the BenchMark system detects 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 59, and 70 HPV genotypes. On the other hand, a low risk probe also detects 6, 11, 42, 43, and 44 HPV genotypes. The INFORM HPV liquid-based prep high-risk probe and the INFORM HPV liquid-based prep low-risk probe are utilized to detect specific HPV genotypes. Lastly, there has been a significant improvement in Pap smear diagnosis. (Lee, J. S. et al; 1997).
Histopathology
Colposcopy and colposcopy-directed biopsy are recommended for patients with abnormal Pap smear findings. 3% acetic acid solution is used in such methods. The samples are observed under 10 to 15-fold magnification after acetic-acid application. Vascular patterns and acetowhitening characteristic of dysplasia or carcinoma are observe under high magnification. Pathologic features of HPV infection like degenerative cytoplasmic vacuolization (koilocytosis) and pithelial hyperplasia (acanthosis) are diagnosed with the help of biopsy. On the other hand, stains have been used to detect HPV antigens or HPV nucleic acids. Polyclonal and monoclonal antibodies are available to detect HPV common antigen. The HPV common antigen is a linear epitope in the middle of the major capsid protein. The antigen is expressed in different HPV subtypes. Peroxidase-antiperoxidase immunocytochemical staining is utilized to detect the bound antibody. (Soma M, & Kamaraj S. 2010). Staining is observed in the nucleus of infected cells. However, in some cases staining is observed in the cytoplasm of koilocytic cells. (Soma M, & Kamaraj S. 2010).
HPV DNA detection
HPV RNA or DNA can be demonstrated in biopsy tissue samples by in situ hybridization methods. In situ hybridization includes probes labeled with radioisotopes or chemically reactive ligands. (Soma M, & Kamaraj S. 2010). The labelled probes are detected by fluorescence or autoradiography. Localization of HPV DNA is possible via in situ hybridization techniques.
Type specific PCR: Sequence variations present in the E6 and E7 genes of HPV subtypes are associated with type-specific PCR. Fourteen type-specific PCRs for high-risk HPV types have been developed recently. The fourteen high risk HPY types are HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68. The specific PCR target approximately 100 bp in the E7 ORF. Depending on the HPV type, the analytical sensitivity of PCR assays is between 10 and 200 HPV copies per sample. Type-specific PCR methods have limited throughput and are currently used in research applications. Type-specific PCR would also utilize multiple PCR amplifications for each sample that can be considered as a major disadvantage of this method. (Soma M, & Kamaraj S. 2010).
General primer PCR: Many research studies have used consensus primers to amplify a broad spectrum of HPV types in PCR. The L1 capsid gene (conserved region of HPV genome) is primarily targeted by most of the consensus primers. A 450-bp fragment within the HPV L1 ORF is targeted by the MY09 plus MY11 primers. The GP5+ plus GP6+ primers target a fragment within the HPVL1 ORF (similar to the MYO9 an MY11 primers). Many methods have been used to identify HPV genotypes after PCR amplification of consensus and general primers. The FDA has approved the Hybrid Capture assay kit for the detection of HPV DNA in cervical cancer samples. The Hybrid capture assay includes two RNA probes, Probe A and Probe B respectively. Probe B pool recognizes high-risk HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68 and probe A pool recognizes low-risk HPV-6, -11, -42, -43, and -44. The DNA-RNA hybrids are immobilized to a microtiter plate. The DNA-RNA hybrids are recognized by a second anti-DNA-RNA antibody conjugated to alkaline phosphatase followed by chemiluminesence and visualization under UV spectrophotometer. Thus, immunohistochemical methods to identify a strain of human papilloma virus (HPV) associated with cervical cancer were discussed efficiently.
References
Soma M, Kamaraj S. Detection of human papillomavirus in cervical gradings by immunohistochemistry and typing of HPV 16 and 18 in high-grades by polymerase chain reaction. J Lab Physicians. 2010;2:31–36.
Lee, J. S. J., P. Wilhelm, L. Kuan, D. G. Ellison, X. Lei, S. Oh, and S. F. Patten. 1997. AutoPap system performance is screening for low prevalence and small cell abnormalities. Acta Cytol. 41:56-64.
Lee, K. R., R. Ashfaq, G. G. Birdsong, M. E. Corkill, K. M. McIntosh, and S. L. Inhorn. 1997. Comparison of conventional Papanicolaou smears and a fluid-based, thin-layer system for cervical cancer screening. Obstet. Gynecol. 90:278-284.
Sheets, E. E., N. M. Constantine, S. Sinisco, B. Dean, and E. S. Cibas. 1995. Colposcopically-directed biopsies provide a basis for comparing the accuracy of ThinPrep and Papanicoloau smears. J. Gynecol. Technol. 1:27-34.
