April 2016, Vol. 5, No. 3
The Rebirth of Ex Vivo Cell Screening for Clinical UseEx Vivo Screening
Interest in high-throughput (HT) ex vivo cell screening of an individual’s cells for personalized cancer treatment is on the upswing, largely due to improvements in technology and assay development. Attempts decades ago to implement the strategy, which assesses the sensitivity and resistance of tumor cells to a panel of drugs and/or drug combinations, were abandoned after it could not be shown to reliably predict a clinical response.1 Advances in sample handling coupled with new genomic information have reinvigorated interest in ex vivo screening methods for drug discovery and as an emerging feature of patient care. Treatments based on ex vivo assays are gaining traction in Europe, where favorable results reported by several groups have suggested new benefits using approved drugs to treat leukemia. These early studies are now being buttressed with expanded trials to establish their predictive value. With that and additional assay development, ex vivo screening should provide new efficacy to personalized medicine.
As a developer of acoustics-based sample handling systems, we at Labcyte have seen the enabling nature of modern HT screening techniques. Acoustic sample handling, in particular, is recognized as a significant technological advance. It uses smaller amounts of biological sample, compounds, or reagents to reduce workflow complexity while reducing costs. Acoustic liquid handling has improved drug potency measurements and data quality that arise from distortions caused by the workflow of other liquid handling procedures,2 especially in the generation of dose-response experiments. As a result, all of the top 10 large pharmaceutical firms use acoustic dispensing in HT screening.
The pharmaceutical industry has experienced a tremendous change in drug discovery with the ability of acoustics to transfer minute volumes of fluids accurately and reproducibly. These improvements are key to efficiently generating ever-increasing amounts of biologically meaningful data, whether on combinations of compounds, defined smaller sets of available cancer therapeutics, or high throughput of a broader array of compounds.
Coupled with robust and scalable data analysis tools, it is now possible to develop large, quantitative data sets of compound-compound interactions. This expands the utility of ex vivo screening. It also increases the understanding of the biological effects of targeted cancer drugs, identifies new synergistic drug combinations, and can be used to select patient populations for clinical development of new therapeutics. The ability to use primary tumor tissues, as opposed to immortalized cell lines, is relatively straightforward in the miniaturized assays supported by acoustic liquid handling.
Our partners at the Institute for Molecular Medicine (FIMM) at the University of Helsinki, Finland, have been at the forefront of this application of HT screening, as shown by recent publications.3-7 As FIMM’s Tea Pemovska and colleagues wrote in Drug Discovery World last summer, “coupling of genomic and molecular profiling of cancer with functional testing of drugs in patient-derived cell samples will make it possible to customize patient treatments as well as to identify biomarker patterns and genetic aberrations that explain the drug responses.”8 Once a responding population is selected, researchers can identify new biomarkers that allow more targeted clinical testing.
The FIMM workflow centers around an HT screening platform that uses acoustic sample handling to determine the best individual treatment for leukemia patients based on ex vivo testing of that patient’s cells. It allows doctors to better determine dosing of leukemia drugs for a specific patient. The FIMM system can assess the sensitivity and resistance of an individual patient’s tumors to hundreds of drugs and drug combinations in a matter of days. With this system, patients in whom several rounds of therapy have failed have received new therapy that led to remission.
This standardized platform and the corresponding drug sensitivity score first developed at FIMM have been used with hundreds of patients’ cancer cells to provide clinicians direction in developing individualized chemotherapy. This ex vivo profiling platform is centered on the robust, accurate, and precise liquid transfer capabilities of acoustic liquid handling and the ability of acoustic liquid handling to provide multiplexing and ultraminiaturization while providing biologically more meaningful results. This platform eliminates errors in assays that result from the loss of materials during standard dilution techniques while significantly reducing overall costs.
The ability to miniaturize and use fewer cells and smaller assays also allows clinicians to look at an array of drugs, including those whose mechanisms may not suggest them as treatment candidates. FIMM and drug giant Pfizer have used the FIMM platform to uncover unanticipated antitumor activity in other drugs. With ex vivo screening, they established that the vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) axitinib selectively inhibited BCR-ABL1–driven leukemias. This unexpected finding shows that comprehensive drug testing of patient-derived cells can identify opportunities for drug repositioning.9 Whereas axitinib was an unexpected active agent in this profiling, other VEGF TKIs were inactive. Since these other drugs have already passed phase 1 and 2 studies, repurposing of these new therapeutic uses can proceed quickly. Repurposing of existing drugs offers significant opportunities for both drug companies and clinicians.
FIMM and others are extending their work into solid tumors. A group at Uppsala University in Sweden has similarly used HT screening in conjunction with a proprietary chemoresistance assay to guide treatment of solid as well as hematologic tumors.10
In Spain, Vivia Biotech runs a centralized, approved diagnostic lab operation focused on developing assays for HT screens that better mimic the tumor microenvironment. In the past, the inability to mimic the native environment sufficiently by accounting for the presence of bone marrow, stroma, platelets, plasma, etc, prevented ex vivo drug sensitivity testing from becoming clinical and predictive. Vivia has profiled samples from patients with acute myeloid leukemia using an automated flow cytometry technique to show how examination of individual tumors can help guide combination therapy.11
Indeed, assay development will be key to further advancing the use of HT screening in the clinic. FIMM has been running a standard cell viability assay, but FIMM and others acknowledge that it is too simple a test—the ultimate challenges are to see what happens in the cells after exposure to a drug, to optimize combinations, and to predict responses. Also, given that the major effect of most cancer chemotherapeutics is to induce apoptosis, a more relevant end point may be to measure total tumor cell death. Nonetheless, the individualized approach pioneered by FIMM is having a significant impact on treatment.
Hospitals that have shown interest in building biobanking capabilities are logical candidates to set up translational medicine programs applying HT screening to individualized treatment. The FIMM project is coordinated with the Finnish Hematology Registry and Biobank, a national program, to biobank samples from all Finnish patients diagnosed with a hematologic malignancy.
In the past, ex vivo screening failed in the clinic for many reasons, including poor precision and accuracy in liquid dispensing and solute loss. Traditional serial dilution techniques can generate errors in drug potency greater than 2 orders of magnitude. Acoustic sample handling is faster, keeps concentrations stable, and reduces compound and reagent usage. The current ability to dispense nanoliter volumes of a compound solution directly to assay plates is revolutionizing compound library design, storage, and distribution, allowing screening of compounds generated by expensive or low-yield syntheses and reducing overall costs.
Part of the past difficulties in extending ex vivo results to the clinic may also be due to the possibility that the in vivo activity of some drugs may not be linked to cellular stress or growth, the most commonly measured ex vivo results. The silver bullet of ex vivo testing, finding a specific genetic mutation that would unequivocally indicate the correct therapy, has failed because the genotypic-phenotypic interactions are far more complex than originally assumed.
Tumors exist in different contexts depending on the individual, and genomics can be overly reductionist because somatic mutations exist in healthy tissue, sometimes at the same level as those in tumors.12 Ultimately, with an initial investment associated with acoustic instrumentation, ex vivo HT screening will make the healthcare delivery system more efficient. If the sensitivity of a tumor to specific drugs or drug combinations is assessed prior to the start of therapy, drugs can be selected that provide the best chance of benefit for a given patient. Conversely, if a drug or drug combination does not work in a reliable ex vivo screen, it would be unlikely to work as therapy. Looking first at an ex vivo assay will both prevent ineffective rounds of chemotherapy and lead to faster cures.
Combining the empirical value of ex vivo HT screening with genomics and mechanism of action–based approaches will greatly enhance individualized, or personalized, medicine.
- Burstein HJ, Mangu PB, Somerfield MR, et al. American Society of Clinical Oncology clinical practice guideline update on the use of chemotherapy sensitivity and resistance assays. J Clin Oncol. 2011;29:3328-3330.
- Ekins S, Olechno J, Williams AJ. Dispensing processes impact apparent biological activity as determined by computational and statistical analyses. PLoS One. 2013;8:e062325.
- Pemovska T, Kontro M, Yadav B, et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discov. 2013;3:1416-1429.
- Pietarinen PO, Pemovska T, Kontro M, et al. Novel drug candidates for blast phase chronic myeloid leukemia from high-throughput drug sensitivity and resistance testing. Blood Cancer J. 2015;5:e309.
- Yadav B, Pemovska T, Szwajda A, et al. Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies. Sci Rep. 2014;4:5193.
- Pietiainen V, Saarela J, von Schantz C, et al. The high throughput biomedicine unit at the Institute for Molecular Medicine Finland: high throughput screening meets precision medicine. Comb Chem High Throughput Screen. 2014;17:377-386.
- Tang J, Karhinen L, Xu T, et al. Target inhibition networks: predicting selective combinations of druggable targets to block cancer survival pathways. PLoS Comput Biol. 2013;9:e1003226.
- Pemovska T, Östling P, Heckman C, et al. Individualised systems medicine: next-generation precision cancer medicine and drug positioning, Drug Discov World. 2015;16:47-53.
- Pemovska T, Johnson E, Kontro M, et al. Axitinib effectively inhibits BCR-ABL1-(T315I) with a distinct binding conformation. Nature. 2015;519:102-105.
- Blom K, Nygren P, Alvarsson J, et al. Ex vivo assessment of drug activity in patient tumor cells as a basis for tailored cancer therapy. J Lab Autom. 2015;21:178-187.
- Bennett TA, Montesinos P, Moscardo F, et al. Pharmacological profiles of acute myeloid leukemia treatments in patient samples by automated flow cytometry: a bridge to individualized medicine. Clin Lymphoma Myeloma Leuk. 2014;14:305-318.
- Martincorena I, Roshan A, Gerstung G, et al. Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin. Science. 2015;348:880-886.
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