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Molecular based testing has become increasingly popular since the recent research progress into the human genome, and what was once used for only inherited disorders is now being used to diagnose neoplasms and infectious disease. (Netto et al 2003). Some molecular based methods in histology have been used routinely for a number of years and these techniques have provided valuable data and information for the patients diagnosis, prognosis and potential therapy for a variety of diseases. Routinely used molecular methods include immunohistochemistry (IHC) and , fluorescence in situ hybridisation (FISH), and most recently polymerase chain reaction (PCR).
Immunohistochemistry was first seen in the 1940's when Coons et al used the direct fluorescent method to prove the presence of pneumococci (Coons et al, 1941). Then in the 1980's the avidin biotin complex method was developed and more importantly it could be used on formalin fixed paraffin embedded sections. This indirect method simply uses labelled antibodies (monoclonal and polyclonal) to detect antigens or proteins within sections of the tissue sample. These antibody-antigen reactions can then be visualised using markers such as chromogen or for the direct technique fluorescence dyes. (www.ihcworld.com)
Diagnostic IHC utilises panels of antibodies rather than using a single antibody, and consists of known positive and negative markers to prove and disprove the presence of certain tumours which can not be identified by a routine haematoxylin and eosin (H&E) stain. These panels are most helpful when dealing with undifferentiated tumours and can narrow down the possibilities of the tumour type if not identify it. (Folpe 1999). IHC stained samples can also be viewed by using standard light microscopy and counter stained with a cytoplasmic dye therefore also viewing the architecture of the sample. (Brandtzaeg 1998) However, IHC does have its disadvantages. These include a lack of specificity making a sample very difficult to interpret with the need to run constant negative and positive controls therefore increasing the workload and also still not guaranteeing the validity of the marker. (www.rndsystems.com). The photobleaching of fluorescent dyes through over exposure to light sources decreases the 'shelf life' of the samples and makes the difficult to store until interpreted by the pathologist. (www.dako.com). Also, a major problem is markers becoming non-exclusive to a particular antigen, this is due to the constant new findings in scientific research bringing previous diagnostic work into question.
However, the advantages of IHC far out weigh the disadvantages and the popularity and importance of the role of IHC to the histopathological diagnosis of patients can be cited in many journals. The graph in figure1 is taken from one such paper and shows the importance and increasing trend in IHC, and how its popularity has risen to become an invaluable diagnostic tool and complimentary method to the conventional H&E. (de Matos et al, 2010)
Figure 1: Shows the number of scientific journals using IHC methods, on the Medline database, between 1960 and 2006. (de Matos et al, 2010).
The most notable roles that IHC plays in the histological diagnosis of a patient include the diagnosing of undifferentiated neoplasms, as previously mentioned this uses specific panels of up to 10 antibodies to narrow down or identify a specific tumour type. The use of a panel can also 'rule out' other suspected tumours giving a more confident diagnosis. (de Matos et al, 2010). IHC can also assist the sub typing of certain neoplasms such as lymphomas. For example, it is valuable to know whether the lymphoma is a B cell or T cell type as each behaves very differently, this subtype would also determine which treatment would be the most effective in halting or regressing the growth or metastasis of the cancer. (www.cancertrialshelp.org). Another important aspect of using IHC staining includes its ability to indicate the possible origin of the primary site for a malignant tumour. As research advances more organ specific antibodies become available to potentially identify the primary site. Examples of antibodies currently available and in current diagnostic use include oestrogen receptor (ER), thyroid transcription factor 1 (TTF-1), progesterone receptor (PGR) and Wilms tumour susceptibility gene 1 (WT-1).
Research papers on the effect of IHC on the histological diagnosis are not common. However, papers such as ' The contribution of immunohistochemical staining in tumour diagnosis' by Leong and Wright, 1987 have studied and validated the role that IHC plays. They have conclude that along side the clinical and other histological laboratory data IHC is an essential tool in the aid of a therapeutic diagnosis. Other papers which validate the data found by Leong and Wright, 1987 include work performed by Schmitt et al 1991, who also concurred that IHC was 'useful for differential diagnosis between carcinoma, lymphoma and melanoma'.
Fluorescence in situ hybridisation
Fluorescence in situ hybridization (FISH) is a molecular method used for localising and detecting specific nucleic acid sequences on a chromosome. This method uses a fluorescent labelled probe designed to target a specific complimentary sequence within the double helix DNA. This is then mixed with the recently denatured DNA, where the probe then hybridises with the sample DNA at a target site, this mixture then anneals back into the DNA double helix, this process can be seen in figure 2. The probe can then be viewed using a fluorescent microscope as seen in figure 3. ( www.genome.gov)
Figure 2: Shows the simplified process of fluorescence in situ hybridisation. (www.serc.carleton.edu)
In a routine laboratory FISH is a method typically used to determine whether there is an amplification of the Her2 gene. FISH is highly sensitive and a precise localisation is achieved from using this method, therefore giving the pathologist the information to make confident diagnosis. This is an important factor in the diagnosing of breast cancer patients as an accurate identification of an over expression would determine the patients treatment. In this particular case, the patient would be eligible for trastuzumab therapy, commomly known as herceptin. This method is of particular importance as it determines a targeted therapy (herceptin) which is very costly. FISH method ensures that the patient will respond to such a drug therefore being cost effective and giving the patient a definitive treatment. (SÃÂ¡ez et al 2006). FISH can also be used for other purposes, examples include the demonstration of cells infected with Epstein Barr virus (EBV) or human papilloma virus (HPV). (Szeles et al 1999). The FISH technique can be quite costly as standardised and validated kits often have to be purchased by laboratories. Also, the test itself is very pH and temperature dependent and requires a highly trained member of staff to over see it. For these reasons, tests (H&E, IHC) are done prior to the FISH technique to determine whether the actual FISH test is required or not.
Figure 3: shows an example case of FISH performed on breast tumour. Red fluorescent probe is her2 and green fluorescent probe is cen-17. A diagnosis is calculated by using the ratio of the two signals. Samples with a her2/cen17 ratio of two or above are classed as positive or over expression. (Royal Liverpool Hospital, sample case)
Polymerase Chain Reaction
PCR is the most recent of diagnostic methods to be used in a diagnostic histopathology lab and has grown in popularity since 1985 when an article in Science was published. (Lo and Feldman, 1994). PCR is used to amplify a single or a few copies of a piece of DNA, generating thousands to millions of copies of a particular DNA sequence. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR which allows for quantitative measurement of DNA or RNA molecules. (Bermingham and Luettich, 2003). PCR also allows for the early detection of diseases such as leukemia or lymphomas. Nearly all B or T cell malignant lymphomas have one or more rearranged antigen receptor genes, therefore this provides a analytical basis for a diagnostic test to assess gene rearrangement or mutation and confirm a diagnosis. (Dis et al, 2012). PCR still has a lot of evolving to do to transmit itself fully into a working diagnostic histology laboratory, and once performed the benefits to a histological diagnosis would be immense. PCR has the ability to show the smallest deletions or mutations indicating the clinical behaviour of a disease, through this deep understanding a disease could be managed much more effectively. In years to come, tumours will be categorised and treated based on the pathways that encourage malignant cell growth and metastasis, than just solely based on the histological appearance of the cells. (Bernard and Wittwer, 2002).
In conclusion, these molecular techniques have been shown to be of relevance in not only the classification and diagnosis but also in the assessing of the patients predicted response to therapy, as shown for the her2 positive patient. The main advantages of molecular biology techniques include a rapid turn-around time which allows patients to start their targeted therapies quickly. They also have high sensitivity and specificity increasing the likelihood of sometimes not only the detection of the disease but also detection of the reason behind the disease, leading to a permanent treatment. However, the main disadvantages include the costly equipment, highly trained staff and reagents which are needed to conduct these tests.
The role of molecular techniques in histological diagnosis is gaining confidence year on year, and there may come a time when test are so specific in their results that only a technician may be required to interpret them. In the meantime, I believe molecular tests in the diagnostic histology laboratory will always follow on from the 'gold standard' Haematoxylin and Eosin stained section. I believe that currently pathologists as a whole are not ready to diagnose a disease on invisible data of molecular tests, and much prefer to see the visible cellular changes under the microscope. However, I am certain that in the coming years histology as a discipline will change beyond recognition, as advancing molecular techniques will take over the role of laborious staining and unnecessary invasive procedures.