Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Researchers Looking for Alternatives to Individual Avatars Have Found Reason to Be Hopeful in Tumor-Cell Based Predictive Models

Mice grafted with human tumors, known as patient-derived xenograft (PDX) mice, have migrated from cancer research labs to the clinic.  But as limitations to modeling patient individual tumors in mice emerge, some investigators are turning to cell-based models and applying new methodologies to support and grow cells in culture.

Conceived by Heinz-Herbert Fiebig and colleagues at the University of Freiburg in the early 1980s, it was hoped that PDX mice would more accurately reflect an individual patient’s tumor in a model system and predict tumor responses to drug therapies.  Dr. Fiebig is the founder and CEO of Oncotest, a company that specializes in preclinical pharmacological contract research.

Since their introduction, commercial labs, including Oncotest, the Jackson Laboratory, and Discovery Group plc Horizon (Horizon), have provided access to a wide range of PDX mice made from donated tumor tissue.  The tissue, cryopreserved for future use after biopsy, serves as the basis for offering drug-testing services to researchers and pharmaceutical companies. Oncotest, for example, says it provides drug-testing services to 16 of the 20 largest pharmaceutical companies, using a library of more than 350 PDX mouse models.

And beyond PDX mouse model libraries for pharma companies, companies now offer individualized avatar mice directly to patients developed using their own tumors.  Champions Oncology provides mouse avatars directly to patients, at a cost of $10,000 to $12,000.  Proponents of these mouse models say they can facilitate the identification of a personalized therapeutic regimen, may prove more useful than genomic analysis, and eliminate the cost and toxicity associated with nontargeted chemotherapeutics.

But formidable barriers impede the widespread use of mouse avatars, scientists say, including the time and expense required to breed and maintain mice engrafted with human tumor tissue.  Development of an individualized avatar takes anywhere from three to six months, more time than some critically ill patients can survive and, in about 30% of cases, Champions points out it hasn’t been able to grow the patient’s tumor in mice.

In a study published in Cancer in April 2014, Justin Stebbing, M.D., Ph.D., and colleagues at Imperial College, London, reported that they worked with Champions to develop avatars with the company’s TumorGraft system for 22 patients with advanced sarcoma. But nine patients died before the results were ready. “Within a couple of months after their surgery or biopsy, they get chemotherapy and they pass away,” says Champions CEO Ronnie Morris. “We build the avatar, but the patient can’t use it.” 

In this study, the scientists said that of implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although several patients died before completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of the 16 (81%) remaining individuals. No patients died during the TumorGraft-predicted therapy.

On the other hand the authors noted that a primary advantage of Champions’ TumorGraft is “that it allows discrimination between the different standard-of-care therapies that may be available, as well as other potential treatments not normally indicated for that tumor.

“Our increased understanding of tumor heterogeneity, even within a single subtype, means that knowing how patients with the same tumor previously responded to a particular drug is no guarantee that the current patient will respond similarly. TumorGraft overcomes this problem by helping guide oncologists to those treatments that are most likely to provide a positive clinical outcome.”


Search for Alternatives

Given the obstacles to using individual avatars to guide patient therapy, researchers in several laboratories are currently looking for alternatives, turning in some cases to tumor-cell based predictive models in a back to the future approach utilizing up-to-date pharmacogenomics and novel cell culture technologies to improve the longstanding odds against success culture of tumor cells from biopsied material.

The team of Jeffrey Engelman, M.D., Ph.D., director of thoracic oncology and molecular therapeutics at Massachusetts General Hospital Cancer Center, has successfully established cell culture models from biopsy samples of lung cancer patients for functional pharmacologic studies. Dr. Engelman noted that while “Genetics has been extremely useful to guiding treatment, in many cases tumor genetics are ambiguous or do not reveal a mutation that informs a therapeutic strategy. These functional pharmacologic studies can identify effective therapeutic choices even when the genetics fail to do so.”  

Dr. Engelman and colleagues described in Science a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome drug resistance. Their cell culture models were derived from patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected to genetic analyses and a pharmacological screen.

With the system they could identify multiple effective drug combinations, they said.  These included the combination of ALK and mitogen-activated protein kinases (MAPK) inhibitors active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation. A combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant-resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, the authors said their strategy could help direct therapeutic choices for individual patients.


Several Approaches

Noting the historical difficulty of coaxing tumor cells obtained from tumor biopsies to grow in culture, Dr. Engelman told GEN that his team typically tries three or four different approaches to optimize the growth of cells from a single biopsy, including 3D culture, organoids, and feeder layers to support the best cancer cell growth.  “We want to get the biopsy to the high-throughput screening phase as quickly as possible and get the results to inform patient therapy as quickly as possible,” he said.

While the application described in their publication involved lung cancer, he notes that his lab is trying the approach on breast cancer, colorectal tumors, and melanoma.  “What’s interesting for us is that there are cancers for which no work has ever been done before,” he noted.

To date, the investigators are “not applying the cell culture technology to the clinic, but are inching closer to doing so,” Dr. Engleman said. “We are confident in the results we get from the screen and believe the data is quite valuable, but we want to make sure there is clinical outcome with therapeutics prior to having a patient enroll in a clinical trial or embark on a specific therapy.”

Dr. Engelman also believes that the technology can be commercialized, but that he is “focused on making it work.” These initial studies demonstrated success in developing NSCLC models NSCLC models in 50% of collected specimens. However, the team believes that success rates could be further improved by using biopsies acquired for specifically for cell line generation.

The authors noted that with their pharmacologic platform, they discovered several previously undescribed combinations in EGFR mutant and ALK-positive lung cancers that were validated in follow-up studies and in vivo.  They speculate that a similar approach could be explored in the future as a diagnostic test to identify therapeutic strategies for individual patients (under the auspices of an IRB-approved protocol).

In their study, they screened the cells after they became fully established cell lines, often requiring two to six months, a time frame that would make this approach less than ideal as a routine diagnostic test. But they say, their results of the program provides the  groundwork for performing screens on viable cells obtained within weeks of a biopsy using newer technologies that would permit screening of the cancer cells while still in the presence of the stroma present in the biopsy.

In a proof of concept study in Nature Methods, investigators working at MGH, Harvard Medical School, the Karolinska Institute, and other institutions showed that circulating tumor cells (CTCs) can be captured in viable form and used to establish cell cultures, potentially bypassing the need for a biopsy as a source of tumor cells to culture.

The investigators captured the CTCs using microchip technology (the Cluster-Chip) developed to capture CTC clusters independently of tumor-specific markers from unprocessed blood.  The device isolates the CTC clusters through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and, the investigators said,  even two-cell clusters can be efficiently captured.

Maheswaran et al., in Cancer Research, used the device to show that the culture of CTCs in the blood of patients with breast cancer enabled them to study patterns of drug susceptibility linked to the genetic context that is unique to an individual tumor.

The investigators established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets.

The authors noted that with optimization of CTC culture conditions, this strategy could help identify the best therapies for individual cancer patients over the course of their disease.

These and other investigators believe, that cell-based methods, once optimized, could bypass the need for whole animal cancer avatars, providing another resource to help inform the choice of therapies likely to be effective in a given patient.








































 

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