Human Papilloma Virus in Lung Cancer Patients

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Question: What steps would you need to take to develop an immunohistochemically method to identify Human Papilloma Virus (HPV) in lung cancer tissue removed from a patient? 


ABSTRACT
Human Papilloma Virus (HPV) is a virus which infects the skin, and particularly moist membranes lining the body. Evidence that HPV may be correlated with the development of lung squamous cell carcinoma came from Syrjane et al 1972, as a result of examining histological samples of oesophageal and cervical cancers, and finding a similarity between the examinations.
Immunohistochemistry (IHC) methods are discussed in this paper, to identify HPV in a lung cancer tissue removed from a patient. The tissue is subjected to multiple processing stages, to ensure the cell morphology, tissue architecture as well as the antigenicity of the HPV epiptopes are maintained and retrieved. Monoclonal mouse primary antibody is used to target the HPV antigen and the signal is amplified via the Labelled Streptavidin Biotin (LSAB) detection system, for visualisation under bright field microscopy. IHC has revolutionised the field of clinical diagnostics as well as medical research, due to being a highly sensitive and specific method, capable of detecting a variety of antigens.

INTRODUCTION
It is estimated that the Human Papilloma Virus (HPV) is directly related to 15-20% of all human cancers (Giuliani, Favalli et al. 2007). With the virus consistently proven to be, not just related to, but the leading cause of carcinomas within the uterus, particularly cervical cancer, where it displays a causality rate approaching 100% (Nour 2009). In other vulnerable areas of the body, more recent scientific research has begun to establish that HPV is implicated in the formation of squamous cell carcinomas of the lungs (Giuliani, Favalli et al. 2007). The first evidence of an association between HPV and lung sqamous cell carcinoma was demonstrated by Syrjane in the early 1980’s where he reported a similarity between the histological characteristics of invasive oesophageal SCC and cervical cancer, suggesting the possibility of HPV aetiology (Syrjanen, Syrjanen et al. 1982).
Further studies have shown the virus capable of interfering with the cell cycle and interrupting tumour suppressive functions, resulting in cell oncogenesis. Oncoproteins E6 and E7, have been strongly linked to interference with the tumour suppressive, p53 and Rb proteins (Prabhu, Jayalekshmi et al. 2012).
With mounting research establishing HPV to be an exceptionally versatile biomarker, and increasing incidences of HPV association with lung cancers, it has become a growing necessity to develop a test for routine HPV virus within lung cancer cells (de Freitas, Gurgel et al. 2016). To develop and implement such a standardised method, a technique that is both highly accurate and cost effective, while also being easily transferable to existing pathology laboratories is required.
Immunohistochemistry (IHC) has become a recognised and adaptable methodology for investigation and diagnostic purposes, utilising antigen-antibody reactions within specific cells or tissues. Once the antigen-antibody binding occurs, the immunohistochemical reaction is demonstrated by the production of a coloured product, which is visible under bright field microscopy (Montero 2003).
This helps to create a technique highly valuable in diagnostic situations, specifically where clinical data and observed morphology under a microscope have proved insufficient to produce a clinical diagnosis (Duraiyan, Govindarajan et al. 2012). Therefore, due to its proven success at identification, through the localisation of tissue antigens, IHC must be considered a highly useful diagnostic tool, when used alongside scientific research. There is no doubt that it would form a solid base for developing a new method to identify HPV within lung cancer tissue.

METHOD

Tissue Processing
Sample Preparation
Initially, the fresh lung carcinoma tissue will be fixed on the tissue slide by applying a fixative such as 10% neutral buffered formalin (Werner, Chott et al. 2000). Next the tissue would need to be embedded in medium such as paraffin, and sections of 4-5um in thickness are cut on a microtome instrument (Grizzle 2009). Fixation is essential for tissue and antigen preservation. The tissue sections must then be mounted on charged slides and dried overnight. This ensures that the section of the tissue with adhere to the slide (Chen, Cho et al. 2010)

Deparaffinization and Rehydration of Tissue Section
To perform antibody staining, paraffin wax must be removed from the tissue sample and the sample must be rehydrated.
• To remove the paraffin wax, immerse the sections in a clearing agent such as xylene for a period of 5 minutes (Williams, Mepham et al. 1997).
• Xylene must be removed by graded washes of xylene and alcohol and the sections are rehydrated, via repeated washes of ethanol with water. For example, subject the tissue sections in 100% ethanol for 10 minutes, and then place them in 95% alcohol for another 10 minutes. In order to complete the rehydration process was the slides in distilled water (dH2O) for a period of 5 minutes (Paulsen, Dimke et al. 2015).
Antigen Retrieval
The next step is to be able to retrieve the antigen epiptopes which have been masked by cross-linking of “methyl bridges” between the proteins in the tissue, due to the fixative, formaldehyde. (Leong, Leong 2007). This can lead to a greater improvement in the detection of antibody staining.
The most efficient epiptope retrieval method, which can be used to retrieve the HPV antigen, is known as the Heat Induced Epiptope Retrieval (HIER) method. This method utilises the heat, which can come from a variety of sources including pressure cooker, autoclaves, or microwaves to retrieve the antigens. Figure 1 demonstrates the process of how antigen retrieval can be performed by using the heat mediated method.
• For example the tissue sections can be subjected to a heat-mediated solution such Citrate buffer in a high heat environment such as in a microwaveable vessel for 20 minutes (Shi, Shi et al. 2011), (Yemelyanova, Gravitt et al. 2013).
Blocking of non-specific background staining
Endogenous peroxidase activity is found naturally occurring in tissues and cells, which can react with the HRP conjugated antibody (Buchwalow, Samoilova et al. 2011). This can result in high non-specific background staining when view under the microscope. Therefore, the tissue must be treated with a blocking agent such as Hydrogen Peroxidase (H2O2), which significantly reduces non-specidinc background staining, resulting to a less likelihood of a false positive outcome (Radulescu, Boenisch 2007).

Amplify the Signal
The staining process that will be discussed in this method is the Labelled Streptavidin Biotin (LSAB) method. The first step would be to inoculate the tissue sections with a primary monoclonal antibody which is specific to the HPV antigen epiptope, such as mouse anti-HPV, followed by the addition of a secondary HRP conjugated Ab, i.e horse anti-HPV (Cardiff, Miller et al. 2014)

An example of such a method is demonstrated below:
• The slides are inoculated serially diluted Anti-HPV, which is a monoclonal mouse anti-HPV protein. Then incubate the slides for 60 minutes at 37oC.
• The next step would be to apply serially diluted horse biotinylated anti-mouse IgG linked Ab to the slides, ensuring that the tissue slice is coated thoroughly. Again the sections must be incubated at room temperature for 30 minutes (Sano, Oyama et al. 1998).

Detection of bound secondary antibodies
In order to detect the secondary Biotinylated anti-mouse IgG, an enzyme conjugated streptavidin can be added to the slides.
• For example apply diluted Streptavidin-Horseradish Peroxidase (HRP) antibody conjugate to the tissue within the wax sections, and then incubate for at least 20 minutes to ensure that all biotin-binding sites on the enzyme conjugated complex are filled (Hofman 2002).

Staining and detection
For HRP-conjugated antibodies, the substrate known as, 3,3’-diaminobenzidine (DAB), can be used, which yields a brown insoluble product.
• For instance a few drops of DAB can be added to the slides and incubated for a period 5 minutes.
• Counter-staining must then is performed using Haematoxylin Mayer, which is a nuclear contrast stain, capable of specifically staining the nuclear chromatin without staining the cytoplasmic organelles, allowing the pathologist to identify to morphology of the cells. The standard factor for a positive reaction is dark brown nucleus of the HPV (Fischer, Jacobson et al. 2008).

Controls
The validity of the IHC results interpretations relies upon appropriate usage of positive and negative controls. Positive controls demonstrate that the antibodies and reagents are functioning properly, whereas negative controls are incorporated in the procedure, to demonstrate that the reaction observed is due to the antigen-antibody complex and not to other exogenous factors, ensuring that results are a true positive (Hewitt, Baskin et al. 2014).

• The positive control would consist of a lung cancer tissue slide treated with the primary mouse anti-HPV, to show specificity of the Ab to the HPV detection. Another tissue slide would be treated with the secondary horse anti-HPV, to demonstrate that the label is specific to the primary mouse anti-HPV.
• The negative control would be a lung cancer tissue slide not incubated with the primary mouse anti-HPV.
• It’s also essential to incorporate a labelling control during the IHC procedure, in order to show that the labelling of the result slides is due to the label added and not to other factors such as endogenous labelling or reaction to other chemical products. A sample of a tissue slide which has undergone all the preparations in detergents and buffers, but has not been exposed to the antibodies, enzyme or the dye would be appropriate as the label control (Burry 2011).

DISCUSSION

An immunohistochemically method was discussed, with the purpose of being able to detect HPV in tissue of a lung cancer patient. The different IHC stages were discussed, including the tissue processing steps, antigen-antibody detection processes, and the staining technique.
Whilst immunohistochemistry is considered a routine procedure, several interdependent factors can affect the outcome result of the immunostaining. It is strongly affected by formalin based fixation, as well as the steps that follow tissue processing in addition to antigen retrieval. Furthermore, the quality as well as specificity of the primary antibody is essential to the final result, as well as the technique of the amplification of the secondary antibody (Otali, Stockard et al. 2009).
Formalin is the gold standard for of fixative used in routine histology, and was the fixative used in the method above. It yields good morphological details of the cells, as well as a consistency in staining. Additionally, it’s highly effective at preventing growth of microorganisms, and being able to stabilise and preserve the tissue for decades (Grizzle 2009).
Antigen retrieval, performed by the direct heat method used in the method above has revolutionised the detection an antigens in tissue which have been subjected to fixatives such as formalin. It can unmask epiptopes of antigens by hydrolysis of methylene cross-links. However a disadvantage of this method is that it frequently results in the damage or even loss of tissue (Shi, Shi et al. 2011).

The antigen staining detection method discussed above was the indirect method, known as labelled streptavidin-biotin method (LSAB). This compromises of three steps, whereby the unlabelled primary antibody against the target antigen is bound with biotinylated secondary antibody. Then this is followed by the addition of biotin-avidin complex, with the HRP enzyme, bound via the biotin. This in turn produces the substrate product DAB, which is a brown colour precipitation (Hofman 2002). Below is a diagram representation of the LSAB

The advantages of the using this method is that it’s more sensitive than the direct method, which compromises of only the primary antibody and the detective marker. This is because in the LSAB method the detection signal is amplified, due to increased number of binding molecules of the secondary antibody to the primary antibody (Cardiff, Miller et al. 2014).

Peroxidase enzyme, is the preferred staining marker among the immunohistochemistry field. Its unique properties with regards to its stability as well as effective staining intensity related to the DAB substrate make it a standard choice for immunostaining (Bussolati, Radulescu 2011). The expectant result of the lung carcinoma tissue would be a brown nuclear staining of the HPV antigen, as demonstrated in figure 3 below.

Fig 3. An illustration of the HPV immunostain positive, in lung cancer tissue (100x magnification) (van Boerdonk, Daniels et al. 2013)

Overall immunohistochemistry biomarkers, such as the HPV antigen can used to assist in the early detection of lung carcinoma, as well as in diagnosis. It can also prove valuable for prognosis of lung carcinoma. IHC has proven as a highly valuable tool for both routine diagnostics as well as in the field of scientific research, and can be a highly effective method to help identify HPV in lung cancer

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