Using DNA Repair Biomarkers to Predict the Response of Cancer Patients to Anticancer Therapy.
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Dr. Christopher N. Parris
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Senior Lecturer
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Brunel Institute of Cancer Genetics and Pharmacogenomics
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Division of Biosciences
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Brunel University
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Kingston Lane
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Uxbridge
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Middlesex, UB8 3PH
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Tel: 01895 266293
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christopher.parris@brunel.ac.uk
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Cytotoxic (cell killing) therapy (chemotherapy and radiotherapy) are the main methods of anticancer therapy for the treatment of early stage (primary) cancers or those that have spread (metastasised) to other sites in the body. The effectiveness of anticancer therapy can be limited by the extent and severity of painful side-effects which are caused by the administration of the treatment. The painful side-effects associated with anticancer therapy can decrease patient welfare, leave the patient with persistent or permanent disabilities and increase patient care cost to the NHS.
Cytotoxic anticancer therapy works by damaging and ultimately destroying the DNA within cancer cells. However, normal non-cancer cells within the body are also destroyed by the treatment and it is the extent of normal cell damage that will govern the level of side-effects experienced by the patient.
Most human cells have number of cellular DNA repair mechanisms that can reverse the effects of DNA damage and return the cell back to its normal condition. Therefore the efficiency of DNA repair in cancer and normal cells will play an important role in:
1. Controlling tumour response and determining the clinical outcome (cure or non-cure of the cancer) by the anticancer therapy.
2. Determining the severity of side-effects experienced by the patient.
Interestingly, some individuals are afflicted by inherited conditions where they have an inborn inability to repair certain types of DNA damage. Such individuals would be at extreme risk of life-threatening side-effects if they were treated with normal anticancer therapy. An example of such a disease is Ataxia Telangiectasia (A-T), in which there is an inability to repair DNA strand breaks caused by radiation exposure. These patients also have an elevated risk of cancer as a result of the disease but it would be lethal to the patient if their cancers were treated with radiotherapy and consequently other treatment options would have to be considered.
While A-T is an extreme case, there is good evidence to suggest that cancer patients with more mild and previously undiagnosed defects in DNA repair mechanisms are also at risk of dramatic and painful side-effects during therapy. In fact our research group recently demonstrated in a patient whom experienced drastic side-effects to radiotherapy (eventually leading to death), a previously un-described defect in a gene controlling the repair of radiotherapy induced DNA damage (Abbaszadeh et al, 2010). Therefore a pre-treatment diagnostic test to determine how cancer patients respond to therapy is likely to prevent such occurrences in the future.
For many years, clinicians and scientists have looked to develop experimental methods that could be used to predict how cancer patients are likely to respond to anticancer therapy. All of these tests rely on taking a sample of tissue (normally a skin biopsy) from the cancer patient. These cells are then grown in culture and exposed to the very same drugs and/or radiation that a patient might receive during the course of the anticancer treatment. The response of the patient’s cells is compared to cells from a normal individual, and if the cancer patient’s cells are abnormally sensitive then it is likely that the patient may experience a high level of painful side-effects. Some of these methods can reliably predict how a patient might respond to therapy, however, the tests take many weeks to perform and therefore cannot provide a result within a useful timescale, since it is imperative to commence therapy as soon as possible following diagnosis. Thus there is a need to develop a simple diagnostic test that will provide useful information to the consulting oncologist within days rather than weeks enabling the design of an effective treatment protocol without delay.
To address this problem our research group has been employing a new method as a predictive test. This method is called the gamma-H2AX assay. When cells are exposed to radiotherapy or anticancer drugs, a break (damage) in the DNA occurs. The cell responds to this damage by activating (phosphorylating) a protein bound to the DNA called H2A. Once this protein is activated it is now called gamma-H2AX and it acts as a “beacon” (foci) to attract the appropriate DNA repair protein to repair the DNA break. If the DNA damage is successfully repaired, the gamma-H2AX foci will disappear within a few hours. If there is a failure to repair (as would be expected in a patient with severe side-effects) then the gamma-H2AX foci will persist. It is possible to measure and quantify the level of gamma-H2AX foci within both cancer and normal cells from patients and this test can be performed within 24 hours.
How the test is performed
- 10 ml of blood is taken from the patient and delivered to the laboratory.
- The white (lymphocyte) cells are purified from whole blood.
- The lymphocytes are treated with radiotherapy or chemotherapeutic drugs to cause DNA damage.
- The measurement of gamma-H2AX foci is performed over the period of one day.
- Results are returned to the oncologist for consideration in designing an appropriate treatment schedule for the patient.
In collaboration with Dr Nick Plowman (Head of Radiotherapy Department) of St. Bartholomew’s Hospital, London, UK, we have recently demonstrated that a group of patients who had earlier experienced severe radiotherapy induced side-effects (including severe nerve damage, deep skin destruction and severe ulceration) during routine treatment were unable to repair radiotherapy-induced DNA damage. Lymphocyte cells were exposed to radiotherapy and failed to repair DNA strand breaks measured by gamma-H2AX levels. Therefore, we have preliminary exciting data using a rapid and convenient assay which suggests we can successfully predict how individual patients might respond to anticancer radiotherapy within the clinical setting.
Experimental Plan
To fully exploit the potential of our diagnostic test we wish to extend these findings to:
- Analyse gamma-H2AX induction in human cells using a panel of cancer chemotherapeutic drugs with different mechanisms of action.
- Exploit our diagnostic test (gamma-H2AX assay) within the clinic to pre-determine painful side-effects in patients with genetic conditions which may leave them at risk of extreme toxicity to radiotherapy and/or chemotherapy.
We have demonstrated that we can use the gamma-H2AX test to predict how patients are likely to respond to radiotherapy. It is now important to take this technology to the next stage and place it within the clinical setting. Initially we aim to test specific groups of cancer patients with:
- A strong family history of cancer, especially breast cancer, as evidence suggests that such patients might be hypersensitive to both radiotherapy and chemotherapy (Moule et al., 2009).
- Potential DNA repair defects which may leave them at extreme risk during radiotherapy.
- Unusual tumours or case histories where the consulting oncologist suspects that there may be an over-reaction to the treatment.
The long-term goal of this research is to provide a mechanism whereby anticancer therapy can be designed for each patient based upon our diagnostic test. Therapy can then be individualised and made more effective and tolerable for each patient.
In conclusion, we have good experimental evidence that using our DNA repair based test, we can identify patients at risk of severe side-effects during therapy. This data has been based upon retrospective studies. However, now we need to move the test forward into a clinical setting. We appreciate that this will take a few years to fully exploit the technology for patient benefit but require funding to perform further experiments towards this goal.
References
- Abbaszadeh F, Clingen PH, Arlett CF, Plowman PN, Bourton EC, Themis M, Makarov EM, Newbold RF, Green MHL, Parris CN. A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a xeroderma pigmentosum patient. J.Med Genet. 2010, 47(3):176-181.
- Bourton EC, Plowman PN, Smith D, Arlett CF, Parris CN. Prolonged expression of the g-H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment. Int. J. Cancer 2011;129(12):2928-34.
- Bourton EC, Plowman PN, Adam Zahir S, Senguloglu GU, Serria H, Bottley G, Parris CN. Multispectral Imaging Flow Cytometry Reveals Distinct Frequencies of g-H2AX Foci in DNA Double Strand Break Repair Defective Human Cell Lines. Cytometry Part A, 2011, Dec 13. doi: 10.1002/cyto.a.21171. [Epub ahead of print].
