Carcinoma of the lungs is one of the common causes of death among adult males and females. There is a great variety of causes involved in the pathogenesis of the disease and among the most frequent ones, is smoking, exposure to pollutants, asbestos and changes in the genome. Furthermore, viral infection and more specifically infection by Human Papilloma Virus, which is one of the main risk factor of the majority of cervical and oropharyngeal cancers, is also considered one of the possible etiologic factors for lung cancer. Immunohistochemistry is one of the methods used to detect Human Pappiloma Virus in specimens taken from patients with lung cancers. Herein; we describe the steps needed to detect Human Pappiloma Virus by using the immunohistochemistry methodology in cancer patients and specifically those suffering from various types of lung cancer.
Introduction
Lung cancer is one of the common and devastating types of malignancies. Approximately 50,000 people per year are diagnosed with the specific type of malignancy in the UK. Primary is the type of cancer which starts from the lungs while secondary is the one which reaches the lung from another site. There are two categories of primary lung cancer according to the type of origin cells. The most common type is the non-small-cell lung cancer which could be squamous cell carcinoma, adenocarcinoma or large-cell carcinoma. The other type is the small-cell lung cancer which is not a frequent type but has a quick rate of metastasis in comparison to the non-small-cell lung type.
The main reason why lung cancer is frequent both among men and women is the increased exposure of both sexes to cigarette smoke (active and passive smoking). Studies has shown that stopping smoking at any age could decrease the risk of death from all pathologic conditions directly related to smoke and lung cancer is one of them. It is obvious that there are other factors which cause the development of the specific pathology with environmental factors, genetic reasons and infections being the most frequent ones. It is estimated that exposure to microbiologic pathogens could cause cancer to 16% of the population. Human papilloma virus (HPV) was found to be involved in the pathogenesis of some type of cancers (cervix, uterus and oropharynx). Interesting enough, it is also involved in the carcinogenesis of the lung. There is a theory that the HPV reaches the lungs via the bloodstream coming from distant infected sites such as the genital system.
HPV belongs to the papilloma viruses, a large family of DNA viruses. According to the character of HPV, which has a high degree of affinity to the squamous epithelium which is similar for the bronchus and lung, it is assumed that HPV is probably related with lung neoplasms.
Immunohistochemistry (ICH) is a technique for staining tissue using antibodies against a particular antigen. The ultimate value of any HPV detection strategy lays in its ability to both recognize the presence of HPV and discern its potential as a driving force of carcinogenesis. An assay may be highly sensitive in its ability to detect trace amounts of HPV, but it may have no clinical value if it cannot discern an incidental virus from an active oncologic agent.. The objective of this essay is to describe the steps needed to identify HPV from a tissue taken by a patient with lung cancer.
Method
Using the right antibodies to target the correct antigens and amplify the signal is crucial for optimal visualization by immunohistochemistry, but it is equally important to prepare the sample correctly in order to maintain the morphology of the cells, the tissue architecture and the antigenicity of target epitopes. Tissue from the target organ is received by various methods like lobectomy, cut or bronchogenic biopsy. It must be rapidly preserved to prevent the breakdown of cellular protein and degradation of the normal tissue architecture. Sample could be simply rinsed to clear from the blood, prior to fixation. The aim is to remove the blood-derived antigens that may interfere with the detection of target antigens.
Most tissue fixatives chemically crosslink proteins and reduce protein solubility, which can mask target antigens during prolonged or improper fixation. Therefore, the right fixation method must be optimized based on the application and the target antigen to be stained. Formaldehyde (formalin), a semi-reversible, covalent crosslinking reagent is usually used for perfusion or immersion fixation. Tissues which are fixed in formaldehyde are typically embedded in paraffin wax to permit sectioning and further processing. It also helps to maintain their natural shape and tissue architecture during long-term storage and to facilitate sectioning prior to IHC.
The formalin fixed tissue should be subjected to section of 4µm in thickness, and then labelled on charged slides. All sections will be dried out by set the slides in a 60 °C oven for 60 minutes. The specimen should be further processed by endogenous peroxidase with 0.3% H2O2 methanol for 30 min followed by three short washes (5 min) in distilled water and then one wash in phosphate buffer saline (PBS).
The paraffin in formalin-fixed, paraffin-embedded (FFPE) sections must be completely removed before IHC staining. If de-paraffinization is not complete, the target antigens will be obscured and the antibodies will be unable to react with them. In fact, paraffin’s hydrophobicity actually repels aqueous solutions containing the IHC staining reagents. Flammable, toxic, and volatile organic solvent xylene has traditionally been used to de-paraffinize FFPE slides, although xylene-free de-waxing alternatives are also available.
Formaldehyde fixation generates methylene bridges that covalently crosslink proteins in tissue samples. These bridges can mask antigen and epitope accessibility and inhibit or prevent antibody binding. As a result, FFPE sections typically require treatment designed to unmask or retrieve the antigenic epitopes prior to staining. This is called epitope or antigen retrieval.
Antigen retrieval is an important step for extended exposure of antigenic epitopes in formalin fixed tissues. Formalin aids in the formation of methylene bridges between adjacent protein molecules. Furthermore, processing of the specimen masks antigens and decreases the sites available for protein demonstration and needs to be reversed in order for the diagnosis to be more precise. Enzyme digestion is one of the types of antigen retrieval which could be used in the case of specimens to detect lung cancer.
Although antibodies show preferential avidity and affinity for specific epitopes, antibodies could also bind without specificity to sites on non-antigen proteins that look similar to the wright binding sites on the target antigen. To reduce the background staining in IHC, prior to staining, the samples are incubated with a buffer that blocks the non-specific sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, BSA (bovine serum albumin), gelatin.
Detecting the target antigen with antibodies is a multi-step process that requires optimization at every level to maximize signal detection. Since the aim is to detect HPV in lung cancer tissue, it would be helpful to add monoclonal antibody anti (HPV) protein. By using the indirect detection method, we check the affinity of proteins to bind biotin to detect a biotinylated antibody that is bound to the target antigen. The antigen-bound antibody is then localized by adding an enzyme-conjugated avidin conjugate which generates an amplified signal when appropriate substrates are added. The use of specific dye like DAB (3,3′-diaminobenzidine) which is oxidized in the presence of peroxidase and hydrogen peroxide, lead to the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity. Finally the use of counterstain will provide contrast to the primary stain and can be cell structure-specific. They are usually added after antibody staining and most common one are hematoxylin and eosin which could be also used in this case. The slide is ready then ready to be seen under the microscope.
Depending on the type of tissue which is under investigation, the use of negative controls could aim in the identifying if the positive signal is genuine. Positive controls could also be used to make sure that antibodies and other reagents are working properly.
The standard positive immunoreaction is dark brown, spontaneous in the nucleus for HPV.
Conclusion
Lung cancer is one of the most common causes of the malignancy related death worldwide and cigarette smoking is the main risk factor. However, only 20% of smokers develop lung cancer, which leads to the conclusions that additional risk factors are involved. The role of HPV as one possible cause of lung cancer was hypothesised as a proportion of morphological changes seen in lung tumours closely resembled those seen in genital HPV lesions. This observation led to intense research in order to identify for the presence of the HPV genome in lung cancer tissue. There are many factors that could influence the association between HPV and lung cancer w like test samples, process methods and detection methods. IHC could be one potentially effective method to identify that connection but it is influenced by a variety of factors like gender, smoking habits, HPV subtype, material, histological type and differentiation, clinical stage. The prevalence of HPV infection in lung cancer patients is quite diverse and there should be further studies in order to identify markers of the infection in the specific type of malignant tissue.
