MICA: Development of a rapid in-vitro multi-drug test device to predict response to combined drug chemotherapy in leukaemia patients, before treatment

This project is to develop a rapid and inexpensive screening device that predicts how a patient will respond to leukaemia chemotherapy within hours. The test will initially be used for Acute Myeloid Leukaemia (AML), a potentially fatal cancer in which white blood cells multiply rapidly and unchecked in the bone marrow and blood. AML patients are usually treated with the anticancer agent cytarabine (Ara-C) in different doses or combined with other agents including Fludarabine, to increase the effectiveness of Ara-C. Up to 40% of patients do not respond to first course of chemotherapy treatment; lack of response is only seen after weeks when a patient may have suffered severe side effects. The nature of AML means it is vital to treat it as soon as possible; a delay of weeks can have severe consequences. This project will develop a quick and simple multi-drug test device based on blood or bone marrow samples BEFORE the start of treatment, to show response to COMBINED drugs used for treatment. The results of the multi-drug test will ensure that a patient receives the right combination of chemotherapy drugs, in the correct dose, to meet their needs and so prevent any delay in effective treatment. The test device will be particularly useful to patients whose leukaemic cells are sensitive to low doses of chemotherapy, allowing them to be treated with minimum drug doses, with less side effects. For elderly patients, where AML incidence and mortality rates are increasing, and where other health conditions may rule out aggressive chemotherapy, this screening test could be particularly timely.
We have previously devised, published and patented details of a rapid test to measure response of AML patient blood or bone marrow samples, within 8 hours, to the SINGLE drug Ara-C. The test uses a bioluminescent bacterial biosensor that has been genetically modified to give increased light output in response to the active form of Ara-C within leukaemic cells; it gives an accurate measure of drug uptake and conversion to the active drug form. We have used our biosensor test on 71 patient samples and shown good agreement with a standard laboratory cytotoxicity test and a test efficiency of 83% when compared with 53 known patient outcomes. The single drug test will be extended to include other commonly used chemotherapeutics and the amount of patient cells per test reaction will be minimised, so that multiple drugs can be tested on a small clinical sample, vital for bone marrow testing, where sample volume is limited. Preliminary studies indicate that our device predicts the effectiveness of combined Ara-C/fludarabine chemotherapy. The test device will be extended to include other relevant drug combinations to exploit its full usefulness for AML treatment.
The main aims of this project are to find the optimum conditions for a multi-drug test device that will predict how a patient responds to clinically relevant doses of combined drug chemotherapy, within 12 hours, using minimal number of patient cells. Also to validate the test device by retrospective testing of 500 clinical samples to correlate biosensor assay results with patient outcomes. This will produce a test device with a simple protocol suitable for routine laboratory use, which will be brought to 'regulatory approval' stage.
The main application of our test device is in AML, to rapidly predict effectiveness of combined drug chemotherapy BEFORE treatment begins. Benefits include determining the most suitable treatment for an individual patient. Those with cytarabine resistant cells can be given effective combined chemotherapy that will enhance survival outcomes; patients with responsive cancer cells can be effectively treated with lower drug dose, reducing acute and long-term side effects including severe infection, infertility, secondary cancer, whilst enhancing quality of life and saving costs of hospitalisation.

This project addresses a need for an in-vitro multi-drug test device that predicts response to combined drug chemotherapy BEFORE treatment, to allow selection of best dose and combination for individual Acute Myeloid Leukaemia (AML) patients. Currently, standard dose aggressive chemotherapy is given at diagnosis but up to 40% of patients do not respond. Over 50% of new AML diagnoses are in the over 60's and prognosis is poor. Co-morbidity and intolerance to side effects are seen with standard dose chemotherapy, giving a need for in-vitro tests to predict response to low dose, less aggressive, chemotherapy.
We have devised a same-day test to measure response of AML blood or bone marrow samples, to the mainstay AML drug, cytarabine. The test predicts patient response with an efficiency of 83%. Our target product is a in-vitro multi-drug test device that predicts response to COMBINED chemotherapeutics. The device uses bioluminescent bacterial biosensors that give a quantitative measure of the active form of chemotherapeutic drugs within leukaemic cells. Drugs such as fludarabine are used in combination, to increase the effectiveness of cytarabine. Preliminary studies show that our device predicts effectiveness of combined cytarabine/fludarabine chemotherapy.
Our technology is accurate, robust and cost effective, with few reagents, suitable for routine laboratory use; end point detection is by low light camera with analysis and interpretation software. The results will inform clinicians quickly on the best treatment options BEFORE patients begin treatment, and reduce subsequent morbidity and mortality.
Our development plan is:
i) optimising biosensor response and test protocols for combined chemotherapy tests
ii) scaling down reagent volume and reducing the number of leukaemic cells per test reaction, to get more tests from a single clinical sample
iii) validating on 500 clinical samples with known outcomes to provide data for regulatory submissions.

Patients with AML and their healthcare teams, including clinicians, laboratory workers and Acute Healthcare Trusts will benefit directly and immediately from this research. Patients will benefit from receiving tailored treatment that will specifically target an individual's cancer. The multi-test device will provide a 'drug response profile' to combinations and doses of chemotherapeutics, to inform patient treatment decision making. Patients with highly responsive cancer cells can be given effective low dose chemotherapy with subsequent reduced side effects, including reduction in hospitalisation, time off work, risk of secondary malignancies and infertility(1). Those with cytarabine resistant cells can be given effective combined chemotherapy(2). The rising numbers of elderly AML patients will be key beneficiaries. These patients may have health conditions that rule out aggressive chemotherapy; test results that indicate effectiveness of low dose chemotherapy will be vital for their treatment.
Patients will also benefit from receiving test results in the first 24 hours of diagnosis, a time of maximum anxiety for patients and their families. Rapid results that indicate the most effective chemotherapy will help with difficult decisions about treatment that have to be made within hours of diagnosis for this rapidly progressive disease. The multi-drug test device will reduce patient anxiety and contribute to improved patient reported outcomes.
Healthcare providers will benefit from targeted chemotherapy leading to reduced hospitalisation of patients with adverse side effects and possible reduction in additional courses of chemotherapy(3). Routine haematology laboratory staff will not require specialised training to obtain results from the in-vitro multi-drug test device(4). Other benefits for haematology laboratories include the relatively low cost of the test device, small volume of test material required, simplicity of assay protocol, reagents with a shelf life of >1 year, lot-to-lot and run-to-run variability of <10% and a sample analyser with a small footprint that can be placed on lab bench-tops for routine use.
In the long term, the bioluminescent biosensor assay technology could be used to predict response to chemotherapy in a range of haematological malignancies, including chronic leukeamias, lymphomas and myelomas and also in solid tumours, where minor adjustments of the assay protocol would allow rapid testing of biopsy material for response to an array of relevant drugs. This is a platform technology; the biosensor is capable of response to a number of cytotoxic drugs including zidovudine(5) used for HIV treatment and could also be used for therapeutic drug monitoring.
The multi-test device has the potential to contribute to the health of patients diagnosed with haematological and solid tumour cancers by rapidly predicting response to different doses and combinations of chemotherapy. It could also be extended for therapeutic drug monitoring in treatment of viral diseases.

1.Redaelli A, Stephens J, Brandt S, Botteman M, Pashos C (2004)'Short- and long-term effects of acute myeloid leukemia on patient health-related quality of life' Cancer Treatment Reviews 30, 103-117
2.Yamamoto S, Yamauchi T, Kawai Y, Takemura H, Kishi S, Yoshida A, Urasaki Y, Iwasaki H, Ueda T.(2007) Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells. Int J Hematol. 85(2):108-15
3.Redaelli A, Botteman M Stephens J, Brandt S, Pasho (2004) 'Economic burden of acute myeloid leukemia:a literature review' Cancer Treatment Reviews 30, 237-247
4.Alloush HM, Anderson E, Martin AD, Ruddock MW, Angell JE, Hill PJ, Mehta P, Smith MA, Smith JG, Salisbury VC. (2010) A bioluminescent microbial biosensor for in vitro pretreatment assessment of cytarabine efficacy in leukemia. Clin Chem. 56(12):1862-70.
5.Patent Application Number PCT/GB2009/00196