Breast Cancer: Gene Expression Methodologies

Study Background

Breast cancer shows wide-ranging profile characteristics concerning the probability of distant recurrence and reaction to treatment. Staging breast cancer by the use of preserved human body cells especially by studying the tumor-node-metastasis offers critical information regarding the risk of distant recurrence and possible mortality as a result of the disease occurrence. However, this method is not very exact in predicting the outcomes, particularly node-negative patients (Paik et al, 2003, p. 16). The tumor node metastasis staging is a method of verifying the extent to which the disease has progressed (stage the disease is at). This is a measure of the degree to which the disease has progressed and is very crucial for guiding the management of the disease as prognosis can be determined from it. This TNM staging system is founded on a retrospective evaluation of several patients who have survived the disease at various stages of prognosis. It’s a reflection of clinical analysis processes and treatments that are used for a specific group of people. Periodic revisions are usually very essential due to the continuing advancement of imaging technology and treatment methods being invented and their survival likelihood.

Why the interest in breast cancer?

Breast cancer prevalence has grown over the past years and it has become one of the leading causes of mortality among women especially in western nations. In the UK, it affects at least one woman out of ten women and its pervasiveness is constantly rising. Even though early diagnosis and better medication are almost guaranteed, many mechanisms that underscore its metastasis ability are not adequately studied or understood. The Breast cancer spreading pattern shows that metastasis of this type of cancer is chiefly through the lymphatic system. This is the reason why the regional lymph nodes are usually the first sites to be affected by when metastasis takes place. From here the cancerous state can spread further to distant tissues in distant metastasis to affect parts of the lungs, bones, or the liver. Even though there are several factors involved in the prognosis of the cancerous states, the regional lymph nodes are the most important factors in breast cancer. It’s been observed that patients found to be having axillary metastasis on prognosis pose the worst prognosis compared to others.

Since there are different markers for the prognosis of the disease, many patients who have expressed node-negative have gotten unnecessary chemotherapeutic treatment due to inadequate prognosis and predictive tests regarding this (Lacroix, 2006, p. 1034). There have been very few studies on lymphangiogenesis in the recent past because there has been not enough information or precise details on the specific lymph markers and growth factors. Currently, these types of obstacles have been overcome by the discovery of a few possible lymphatic-specific markers (Paik et al, 2003, p. 16). The relationship between the spreading of breast cancer through the lymph system and evidence of distant metastasis was not formerly studied in human beings (Lacroix, 2006, p. 1034). By the use of the lymph markers, it has been made possible now to investigate the relationship between the spreading of cancer through the lymph and distant cancer metastasis. Studies have used lymphangiogenesis by studying the relationship between tumor nodes and recurrence of the disease (Weissenbacher, et al, 2010., p. 28).

With the increased study of the occurrence of cancer at molecular levels, there is the likelihood of a greater understanding of the cancer recurrence risk and its response to medication (Lacroix, 2006, p. 1036). When cancer therapy is being planned, assays for estrogen, progesterone and HER2 receptors are habitually done to assess the statuses of these receptors (Gruvberger et al, 2001, p. 5981). Several markers have been studied but only a few putative markers are clinically authenticated. Two new matchings progress allow a new advancement and clinical justification of prognosis and prognostic markers (Lacroix, 2006, p. 1037). The first is to use gene expression assays. This is where the preserved tissue containing numerous genes in a frozen state is analyzed by DNA microarrays. This is done for the identification of markers for their validation. Even though frozen oncogenic tissues are available for studies, very few stored in tissue banks are capable of initiating long-term recurrence. Second, the polymerase chain reaction can be used for quantifying gene expression hence the Reverse transcriptase PCR can validate genes that express tumors for clinical study (Cobleigh et al, 2005, p. 8624).

To establish a responsive, precise, reproducible, and workable test for breast cancer prognosis, Cobleigh et al have established and optimized a multi-gene gene RT-PCR expression assay (Cobleigh et al, 2005, p. 8624). This assay is being used for measuring the expression of about 187 genes associated with the occurrence of cancer as well as five reference genes.

Evaluation of the Gene Expression Methodologies

Tumour-node-metastasis (TNM)

For the categorization of breast cancer, the use of TNM staging has been developed to become the standard system over the past 50 years. However, over that duration, there have been a lot of changes that have taken place and in 2003 a new TNM system was created (Weissenbacher, et al, 2010., p. 28). The prognostic relevance of the T (for Tumour) category is now a constant factor in the current and even previous means of classification (Cobleigh et al, 2005, p. 8624). survival of patients relies on two different types of prognosis; the size of the tumor as a marker of the disease and as a factor dependent on time and the physiological factors like histological grade which reflects the aggressiveness of the tumor (Lacroix, 2006, p. 1037). Other factors affecting prognosis include the major female hormones estrogen and progesterone receptors and the number of mitotic divisions represented as MF/10HPF (XX per ten high power fields) (Gruvberger et al, 2001, p. 5981). The treatment follows the suggestions from professionals around the world even as the TNM profile changes. The TNM factors are set for categorization and as mentioned earlier several changes have been made to this system to ensure efficiency (Cobleigh et al, 2005, p. 8624). However, the most important issues to look at are the basics for diagnosis:

T – Tumour stage

TX – this is when the primary tumor cannot be analyzed (AJCC, 2010, p. 348)

T0 – this is when evidence of a primary tumor cannot be assessed

This – this is when there is carcinoma present- could be either Paget’s disease

T1 – this is when a tumor of up to 2 cm can be diagnosed
T2 – this is the stage when the tumor is between 2 and 5 cm
T3 – this is when the tumor exceed 5 cm (AJCC, 2010, p. 348)
T4 – this represents a tumor that grows on the skin or chest wall despite the size

For the assessment of the node, the following classification is used to rate cancer;

NX – This level is when the regional lymph nodes affected cannot be evaluated
N0 – this is the state when cancer cannot spread to the regional lymph nodes
N1 – this is when breast cancer can spread to nodes on the same side as the breast for 1 up to 3 axillary lymph nodes (Weissenbacher, et al, 2010., p. 29)
N2 – this is when cancer breast to lymph on the same side as the affected breast for up to 9 axillary nodes (AJCC, 2010, p. 348)
N3 – when the breast cancer is said to have moved to over 10 axillary lymph nodes or when it has spread to lymph beyond the collarbone

To assess metastasis, the following categories are used to classify distant metastasis usually denoted as M- stage

MX – This is the stage when the presence of distant metastasis cannot be assessed
M0 – this is when there is no diagnosis of distant spread (AJCC, 2010, p. 348)
M1 – this is when cancer spread to distant body parts is diagnosed.

Once the TNM groups have been established, the information is combined for stage grouping. This is because cancers at the same stage tend to present a similar outlook and hence are managed the same way. The stages are expressed in Roman numeral as in stage I (Weissenbacher, et al, 2010., p. 28). For example, stage 0 is Tis, N0, M0 which is the earliest cancer of the breast also referred to as ductal carcinoma this can then proceed to the next stage of Lobular carcinoma which is now the true cancer of the breast. The stages are from stage I to IV (AJCC, 2010, p. 348).

DNA Microarrays

Differentially expressed genes are those that have a very different way of expressing in two microarray experiments. This technique can be used for the identification of disease markers relevant to clinical diagnosis by use of techniques of gene expression in a faster and systematic manner (Severgnini et al, 2006, p. 43). The basic idea of the experiment for verification of the ability of that specific gene to notably expresses differently in two different groups. The test is based on statistical hypothesis testing which is critical for comparing several pieces.

Cancer develops when there are DNA alterations which come as mutations. Because these mutations are commonly as a result of duplication of bases or deletion, the resulting DNA is usually longer or shorter and this results in dramatic changes in the affected cells like the tumour cells (Severgnini et al, 2006, p. 43). Cataloguing such gens has lead to better understanding of the disease and hence guided better development of therapy. Herceptin treats cancer where HER-2/neu receptors are in excess. The hypothesis is that some genes have possible specific markers for cancer (Sorlie, et al, 2003, p. 8419). The DNA array is a slide with numerous genes profiles arranged in rows and columns ready for comparisons by mapping the changes to normal genes.

RT-PCR is the most sensitive method for detecting mRNA in the current world. The process is so sensitive that it can be used to quantify RNA from a single cell. The process allows exponential amplification of DNA. The process involves use of DNA primers of about 20 nucleotides (Severgnini et al, 2006, p. 43). The nucleotides complement each other and the primers are usually extended by nucleus enzyme (the DNA polymerase enzyme) so that extension of the required sequence is produced. The primers can be used for making more copies of DNA strand (Estebanj et, 2003, p. 850). Even for cells fixed in formalin can be expressed hence RTC-PCR is used for validating the genes responsible cancer recurrence in clinical research Cobleigh et al used Taqman RT-PCR for measuring small DNA pieces from the preserved blocks of tissue (Cobleigh et al, 2005, p. 8626). The specific genes were selected after a study of breast cancer literature on prognosis especially characteristics like metastasis, proliferation, angiogenesis and oncogenesis.

Contribution to Current Understanding of Cancer

This paper has contributed to the current knowledge in breast cancer management especially the use of gene expression methodologies. Basically gene expression describes the way specific genes are behaving in terms of the number of times that gene is expressed, how that gene is transcribed and how it encodes specific proteins (Severgnini et al, 2006, p. 43). The process includes steps like transcription which copies the DNA information to RNA through the mRNA. Modern technology enables researchers to count the number mRNA molecules found in any tissue as this helps to estimate the number of consequent proteins (Van’t Veer et al, 2002, p. 531). This paper has also highlighted high-throughput methodologies like use of the gene expression technologies DNA microarray and RT- polymerase chain reaction in assessment of breast cancer recurrence and metastasis ability. These methods allow simultaneous counting of the number of transcriptions made and it contributes to the tissue’s global activity (Sorlie, et al, 2003, p. 8419).

Sampling of the tumour tissues of breast cancer is usually done during diagnosis process and the tissues are preserved for further analyses in well equipped laboratories (Estebanj et, 2003, p. 850). Pathologic review here is done for assessing the cancerous cells. This process is then followed preparation of RNA and integrity assessment. Previous studies have shown that suitable sampling can be used to quantify the RNA level, hence enabling evaluation of gene expression (Sorlie, et al, 2003, p. 8420). This means that the high-tech gene expression assessment targets quantification of mRNA in a particular cell. From such technology and the knowledge of various diseases like cancer enabled creation of tumour gene expression profiles (Cobleigh et al, 2005, p. 8627). The profiles allow verification of occurrence of certain genes.

Because there is still little understanding of the role of genes in metastasis or recurrence of cancer this paper provided gene expression profiles that can be used for diagnosis and prognosis of cancers. Sometimes it has been unclear which clinical characteristics or descriptions of tumours are being assessed to enable drawing conclusion on for deciding the type of therapy to be given (Cobleigh et al, 2005, p. 8627). Furthermore, this also provides information on the type of reaction or response that the disease condition will be likely to give. Characteristics like the aggressiveness of a tumour, the ability of cancer to metastasize, and the general responsiveness to therapy be it chemotherapy, hormone therapy or radiation. Each of the therapies may be involving a totally different gene directing prognosis. Nonetheless, this study has shown that a good health research analyses all aspects concerning the disease under study (Cobleigh et al, 2005, p. 8627). This means characteristics under assessment in a particular study should be regularly deduced from the medical profiles, clinical characteristics and the target populations. We can understand that results from heterogeneous populations that are diverse clinically and therapeutically may not be best for determining prognosis and the risk that a certain cancer poses to the patient.

The researchers used the DNA microarrays to contribute to the improved clinical diagnosis of cancer (Van’t Veer et al, 2002, p. 531). This method is also useful in assigning tumours to the already know classes of cancers and have been profiled on the databases. It’s been indicated that DNA microarrays are finding increased use in selection, evaluation and will also find special use in pharmaceutical development in future as the results will be able to direct clinicians to the causes of the cancer. Based on the results and conclusion drawn, the DNA microarrays will play a big role in diagnosis and also in monitoring the results of the therapy used. Other studies have also shown that gene expression assessment by use of DNA microarrays was very successful in the study of leukaemia. These studies provide an algorithm for that allows classification of various leukaemia’s, hence differentiating the acute lymphoblastic from myeloid leukaemia. A number of approaches used for diagnosing new types of cancers or assigning them to the existing classes have also been reported.

Recently, the comprehensive assessment and exploration of gene expression technologies have been conducted greatly to discover the genes that appreciably impact on clinical outcomes. This method of DNA microarray will definitely be among the most effective instruments for comprehensive assessment of gene expression (Van’t Veer et al, 2002, p. 531). Numerous studies have also been carried out on breast cancers and have revealed a number of crucial facts clinically (Cobleigh et al, 2005, p. 8627). Nonetheless, the technical hitches of this method in clinical application like accuracy, expenses, data processing and reproducibility have been impacting on it negatively. Downsizing the DNA microarray is a way to conquer these obstacles. Estrogen and progesterone receptors have been identified as among the most significant clinical targets when studying prognosis of breast cancer. However, the signalling of oestrogen is still not vividly comprehended (Gruvberger et al, 2001, p. 5981). DNA microarrays for comprehensive gene expression is potentially useful for assessing the signalling cascade, particularly for evaluating physiological events in the cells triggered by stimuli like oestrogen.

Breast cancers demonstrate a very widely varying characteristics concerning to the probability of recurrence and the disease’s response to medication. Using tumour-node- metastasis to Stage human breast cancer provided relevant data concerning the risk of distant recurrence and even probability of death. Still this is not precise particularly the node negative (Estebanj et, 2003, p. 850). From these results, doctors tend to give the node-patients chemotherapy that is uncalled for due to lack of predictive tests.

The TNM system is not a perfect system of study but it is just a representation of the existing efforts of offering clinically useful information and reflection of the current data. There have been suggestions that TNM system should be improved so the estimation of prognosis would be more precise (AJCC, 2010, p. 353). An important factor of better staging will standardise the process of collecting important information that will influence treatment. The most important change in this group is in the decision making time and it’s at this stage that the micro-metastases are isolated from normal cells based on their size. Micro-metastases are small lesions of less than 2 mm and can be categorised clinically.

Even though there have been great developments in isolation of tumour cells and micro-metastases particularly those that present nodal metastasis, many researchers are shifting to venture in other possible significance of tumour cells deposited in bone marrow (Sorlie, et al, 2003, p. 8421). It was very enticing to purport that some of the markers would direct the study to something that TNM does not currently offer – the exact prognosis of individual cancers.

Other Approaches Addressing the Research Question

Staging is the extent to which the spread of cancer can be assessed. This is based on factors like invasiveness, tumour size and the number of lymph node affected following diagnosis (Suri & Rangayyan, 2006, p. 430). From this study the conclusions drawn show that the archives provided crucial information concerning the risk of distant recurrence in cancer patient with over 10 lymph nodes affected. The stage of cancer is important for prognosis and for determining therapy options. Staging is discovering how cancer is spread in the body. There are several methods that can be used for staging so that one can use the data to identify the best therapy.

These are imaging tests that can be performed following diagnosis. Chest X-ray is done to evaluate possible spread of the cancer to the lung tissues. Mammogram is done to identify other areas of the breast that could be having cancerous cells (Suri & Rangayyan, 2006, p. 430). A bone scan on the other hand enables assessment of the bones for possible metastasis. This system can be more helpful than X-Rays since it can assess every bone at once. Computer tomography scan is just like X-ray but it takes numerous pictures around the patients and then combined these pictures to produce a 3 – dimensional object (Suri & Rangayyan, 2006, p. 434). Magnetic resonance imaging is also used to examine the breast. Waves of sound can also be used to search for cancerous cells providing a picture of how the spread pattern has progressed. The use of sound for imaging is the ultra sound technology.

Reference List

AJCC (American Joint Committee on Cancer), 2010. Cancer Staging Manual, 7th Ed, Edge, SB, Byrd, DR, Compton, CC, et al (Eds), New York, Springer-Verlag, pp. 347-377.

Cobleigh, M.A., et al. 2005. Tumor Gene Expression and Prognosis In Breast Cancer Patients With 10 Or more Positive Lymph Nodes, Clin Cancer Res; 11 (24), pp. 8623-8630

Estebanj, B., et al. 2003. Tumor Gene Expression And Prognosis In Breast Cancer: Multi-Gene RT-PCR Assay Of Paraffin-Embedded Tissue. Proc Am Soc Clin Oncol, Issue 22 pp. 850.

Gruvberger, S., et al. 2001. Estrogen Receptor Status in Breast Cancer Is Associated With Remarkably Distinct Gene Expression Patterns, Cancer Research Journal, Vol. 61, Issue 16, pp. 5979 – 84.

Lacroix, M. 2006. Significance, Detection and Markers of Disseminated Breast Cancer Cells, Endocr Relat Cancer; 13, pp. 1033–1067

Paik, S., et al, 2003. Multi-Gene RT-PCR Assay for Predicting Recurrence in Node Negative Breast Cancer Patients-NSABP Studies B-20 and B-14. Breast Cancer Res Treat, 82, p. 16.

Severgnini, M., et al. 2006. Strategies for Comparing Gene Expression Profiles from Different Microarray Platforms: Application to a Case-Control Experiment, Anal Biochem. 353: 43

Sorlie, T., et al. 2003. Repeated Observation of Breast Tumor Subtypes in Independent Gene Expression Data Sets, Proc Natl Acad Sci USA, 100: 8418–8423

Suri, J.S & Rangayyan, R.M. 2006. Recent Advances in Breast Imaging, Mammography, and Computer-Aided Diagnosis of Breast Cancer, Bellingham, WA; SPIE Press, pp. 430-453

Van’t Veer, L.J., et al. 2002. Gene Expression Profiling Predicts Clinical Outcome Of Breast Cancer. Nature, 415: 530 – 6

Weissenbacher, T.M., et al. 2010. Multicentric And Multifocal Versus Unifocal Breast Cancer: Is The Tumor-Node-Metastasis Classification Justified? Breast Cancer Res Treat, 122: 27–34

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