Human Breast Cancer: Correlation Of Relapse And Survival With Amplification Of The HER-2/neu Oncogene

Background

Discuss about the Magnetic Particles For The Separation Purification.

The given study titled, “Human Breast Cancer: Correlation of Relapse and Survival with Amplification of the HER-2lneu Oncogene”, aimed to identify the role of genes in the biological behaviour or pathogenesis of human breast cancer. During the period in which the given study was undertaken the evidences relating the proto-oncogenes with the induction or maintenance of human malignancies continued to arise. This data linking the proto-oncogenes to cell growth constituted the expression of these genes in response to proliferation signal. For the study purpose 189 primary human breast cancers were investigated through amplification of HER-2 gene. The subjugation of this particular gene comes from the fact that it was one of the 3 proto-oncogenes showing direct evidence of being related to growth factor receptor, tyrosine kinase. The HER-2/neu gene also shows close relation with gene for epidermal growth factor. Therefore, the study was conducted to highlight the role of this gene in causing primary breast cancers in humans, as indicated by the amplification of HER-2/neu gene in human mammary carcinoma cell line. With the help of results obtained from the present study the researcher’s aimed to show the correlation of the respective gene with disease relapse and overall survival.

The initial survey consisted of 103 tumours. The individual tumours were subjected to Southern blot analysis and the degree of amplification was evaluated using dilutional analysis, and soft laser densitometry scanning. However, the results from this initial analysis did not show any correlation between gene amplification, and status of estrogen receptor, status of progesterone receptor, tumour size, and the age at diagnosis. However, the researchers noticed a trend for association between HER-2/neu amplification and number of positive lymph nodes in the patient. The multivariate regression analysis showed the number of positive nodes to be the only significant factor to correlate with HER-2/neu amplification. These results indicated towards the possibility of discriminating between the node positive patients on the basis of amplification of HER-2/neu gene. To confirm the results of preliminary survey another study consisting of 86 samples from patients with positive axillary nodes, and the data for survival and relapse was conducted. The results from this confirmed the notion that lymph nodes and HER-2/neu amplification shares significant relationship, and thus could possess sufficient prognostic value. The univariate survival analysis comparing amplification information with relapse and survival was performed. The patients who had more than 5 copies of the gene showed shorter disease free survival times, with respect to patients having no amplification. Thus, the study indicated towards the existence of relationship between amplification of proto-oncogenes and tumour progression in specific cancers.

Study Methods

The below mentioned techniques of molecular biology were used in the given paper:

1. DNA extraction: The DNA extraction method followed in the given study was phenol detergent method. The treatment with detergent is an effective yet gentle method for disrupting the cell membranes, resulting in irreversible denaturation. The Sodium Docdecyl Sulphate detergent was utilized, which helps in denaturation of chromosomal DNA of high molecular weight. Phenol on the other hand helps in removal of debris such as proteins, lipids, carbohydrates, and other cellular debris. This method of extraction is reliable, cheap, and completely removes proteins. However, the weakness of the technique lies in the reliability issues. The technique has to be used with high degree of precision and accuracy, otherwise the DNA can get damaged and contaminated.

1. Digestion with EcoRI: EcoRI is a restriction enzyme which was employed to breakdown the isolated DNA into smaller fragments. The advantage of using EcoRI or any restriction enzyme with known restriction sites could help in obtaining the desired number of bands. This allows for the selective evaluation of the bands to observe the desired traits or results by radiolabelling them and resolving using electrophoresis. Also, as the restriction sites are known, the cuts made by them are predictable and consistent. However, the disadvantage of the technique is that it may result in generation of large number of fragments, and therefore generation of patterns which might not be easy to comprehend and study.

1. Agarose gel electrophoresis: The digested DNA was separated on agarose gel by electrophoresis, to separate the DNA molecules by size. It is achieved by allowing the fragments to run under electric field, wherein the charged nucleic acid molecules run on matrix of agarose gel. The technique allows easy processing of the DNA molecules, which could be recovered from the agarose gel at the end of process by melting the gel or digesting it by using enzymes. However, this technique is low throughput, and requires substantial amounts of starting sample. It shows poor separation of samples of low molecular weight. Also, the gel is susceptible to melting, due to which the genetic material may adopt shapes which are not needed.

1. Southern blot analysis: The electrophorised DNA fragments were transferred to nylon membrane, hybridized with 32P labelled HER-2/neu-1 probe known to detect a 12-kb hybridizing band in human DNA. The nylon membranes themselves possess the advantage of being less fragile, resistant to handling damage, and can efficiently bind DNA as small as 50bp in length. This technique allows for the quantification of digested and indigested DNA molecules, helps in analysis of repetitive sequences by a single probe. However, this technique also requires extraction of large amounts of DNA, and is capable of analysing only limited CpG sites.

1. Dilution analysis: This technique allows the quantification of cells possessing observable functional activities. Besides the quantification of cells, it also allows the analysis of regulatory processes which are underlying the observed cellular activity. However, the initial screening of the cells proves to be a tedious task.

In the light of review of techniques of molecular biology in the given paper, it was observed that the different purposes could be solved using better and latest techniques of molecular biology available presently. Therefore, the following proposal with improvements is suggested:

DNA extraction: The three basic steps involved in DNA extraction involve lysing the cells, separating the DNA from cellular components, and isolation of DNA. The phenol extraction method requires high degree of precision, is time consuming, and risk of DNA contamination is high. Therefore, here the improved method of solid phase extraction method, utilizing magnetic beads is suggested. A number of kits are available in the market, such as  Applied Biosystems’ PrepFiler, Promega’s DNA IQ, and Qiagen’s QIAamp kits (Elkins 2013). This method is based on the interactions between the selected analyte in liquid phase and supporting material. The target analyte is separated from the solution on the basis of specific hydrophobic, polar or ionic properties of both the solute and the sorbent. The nucleic acids are separated from the mixture on the basis of complementary hybridization (Berensmeier 2006). Being economical, it also ensures high degree of recovery, as the DNA binds to the paramagnetic or silica beads. Also, the use of centrifuge is not required which can often break the DNA due to shearing force, therefore longer strands of purified DNA are obtained (Ali et al. 2017).

As the detection of HER-2 copy number is a strong indicator for progression of tumour therefore it is extremely necessary to accurately measure the HER-2 copy number. The given paper presented the techniques of cloning and southern blot hybridization to detect and quantify HER-2 gene amplification. The degree of amplification was detected using dilution analysis and soft laser densitometry scanning. However, these procedures prove to be time consuming and cumbersome. In order to achieve highly sensitive, and precise results with low degree of subjective alteration, an advance technique of droplet digital PCR is suggested. This technique has been used by previous researchers to detect the copy number of HER-2 for prognostic measures (Wang et al. 2017; Belgrader et al. 2013). The droplet digital PCR helps overcome the issues of scalability and practical technologies required for effective implementation of digital PCR technique. This method is based on the fractionation of the sample into 20,000 droplets, wherein the PCR amplification of the template molecules occurs in each droplet, using the TaqMan assay. ddPCR tends to improve upon the immunohistochemical and FISH methods of detection of HER-2 copy numbers, as it is able to quantify DNA and RNA targets with high level of precision and accuracy (Heredia, N.J. et al. 2013). The researcher needs to obtain suitable assay kit from the manufacturer, which can facilitate the detection of copy number variation. The drawback of this technique is its high cost, and demand for consumable materials. Also, multiple steps of dilution, transferring, and pipetting are required which might increase the hands on time (Liang 2015).

Both the techniques proposed here do not suffer from any substantial drawbacks or weaknesses, and thus pose as quite suitable for measurement of copy number variation, which requires high precision and sensitivity.

References

Elkins, K.M., 2013. Forensic DNA biology: a laboratory manual. Academic Press.

Berensmeier, S., 2006. Magnetic particles for the separation and purification of nucleic acids. Applied microbiology and biotechnology, 73(3), pp.495-504.

Ali, N., Rampazzo, R.D.C.P., Costa, A.D.T. and Krieger, M.A., 2017. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. BioMed research international, 2017.

Wang, Y., Tsang, J.Y., Cui, Y., Cui, J., Lin, Y., Zhao, S., Law, P.T., Cheung, S.Y., Ng, E.K., Gary, M.K. and Ke, Z., 2017. Robust and accurate digital measurement for HER2 amplification in HER2 equivocal breast cancer diagnosis. Scientific reports, 7(1), p.6752.

Belgrader, P., Tanner, S.C., Regan, J.F., Koehler, R., Hindson, B.J. and Brown, A.S., 2013. Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue. Clinical chemistry, 59(6), pp.991-994.

Heredia, N.J., Belgrader, P., Wang, S., Koehler, R., Regan, J., Cosman, A.M., Saxonov, S., Hindson, B., Tanner, S.C., Brown, A.S. and Karlin-Neumann, G., 2013. Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods, 59(1), pp.S20-S23.

Liang, S., 2015. Detection of MegaTAL-induced HIV pol Mutation By Droplet Digital PCR (Doctoral dissertation).