October 1, 2015 (Vol. 35, No. 17)
Kyra Mumford Ph.D. R&D Manager Angle
Circulating Tumor Cell Isolation Platform Boosts Versatility in Molecular Analysis
In 1971, President Nixon signed the National Cancer Act, declaring a War Against Cancer. Over the past 30 years, there has been an explosion in molecular oncology with consequent clinical approval of anticancer drugs targeted at specific molecules. The power of drugs, such as trastuzumab, bevacizumab, and gefitinib has resulted in substantially increased patient survival. However, the impact of these drugs continues to be restrained.
Tumor heterogeneity and cancer evolution lead to high relapse rates, and tumors that initially show response frequently develop resistance to initial therapies. Consequently, a major unmet clinical need today is the temporal molecular characterization of cancer. Clinicians require new methods to deploy molecular techniques in initial diagnosis and niche characterization of tumors and for ongoing monitoring of cancer evolution, drug resistance, and relapse. This requires noninvasive methods of biopsy to enable repeated examination of patients to determine the on-going evolution and the progression of their disease.
Blood samples have long been convenient for disease diagnosis. A key factor that makes blood-based diagnosis possible is the presence in blood plasma of proteins and other molecules whose presence indicates disease states. Molecules used in cancer diagnosis include CA125 for ovarian cancer, PSA for prostate cancer, and alpha-fetoprotein (AFP) for liver cancer. The limitation of blood-based diagnosis is the availability of disease-specific molecules.
Yet, a huge number of biological molecules and processes have the potential to enable new diagnostics, and as the number of known linkages between biomolecules, cellular processes, and disease increases, so will the precision of clinical understanding. In other words, access to the full portfolio of molecular changes that participate in disease, be they modifications in small molecules, proteins, DNA, or RNA, will enable precision medicine.
Circulating Tumor Cells
Recently, the ability to capture cell-free DNA (cfDNA) from a blood sample for genetic analysis has broadened the potential to diagnose and prescribe targeted treatments for cancer. During cancer progression, cells break off from the primary tumor and move into the peripheral circulation, where they play a role in metastasis. These circulating tumor cells (CTCs) provide a source of cancer cells in blood samples and the advent of the CellSearch® Circulating Tumor Cell Kit in 2004 successfully established the clinical utility of CTCs.
CellSearch generated a significant expansion of CTC research, and the product is FDA certified for a cancer prognosis analysis based on enumeration of CTCs in cancer patient blood. However, to progress CTC use for precision medicine requires their isolation in a form enabling versatility in their molecular analysis.
Angle’s Parsortix CTC capture system is a microfluidic device that was developed to provide CTCs suitable for use in precision medicine applications (Figure 1). Several aspects of its design provide the required versatility in molecular analysis of cells subsequent to their capture from blood:
- The isolation mechanism is epitope independent
- Isolated CTCs are highly enriched, with a small quantity of residual blood cells remaining
- Cells are harvested as free suspensions in small volumes of buffer.
To date Parsortix has successfully isolated ovarian cancer CTCs. Figure 2 shows a cell stained for p53, an accepted marker for high-grade serous ovarian cancer. In addition, Dr. Yong-Jie Lu at Barts Cancer Institute, Queen Mary, London, has used the instrument to isolate CTCs from prostate cancer patients. By using both cytokeratin and vimentin markers, Dr. Lu has quantified cells in differing stages of the epithelial-mesenchymal transition. His research group successfully harvested CTCs from a cohort of 52 prostate cancer patients, and correlated cytokeratin negative, vimentin positive, and CD45 negative circulating cells with levels of PSA in blood.
Strikingly, mesenchymal circulating cells were statistically significantly higher in patients with greater PSA. “This work has confirmed the ability of the Parsortix system to harvest clinically relevant mesenchymal cells and that these cells correlate with the patient PSA level, the current gold standard for assessing whether the patient’s prostate cancer is progressing,” Dr. Lu commented.
In addition to protein characterization, Parsortix has also enabled the discovery of clinically useful information, by RNA analysis of harvested cells. A research team led by Prof. Robert Zeillinger, head of the molecular oncology group, and Dr. Eva Obermayr, principle investigator at the Medical University of Vienna, used a gene expression panel which showed 100% specificity and sensitivity in detecting ovarian cancer in peripheral blood when using Parsortix.
“The Parsortix technology contributes to the unprecedented specificity of the overall approach, by providing a high-purity CTC sample. Parsortix is a label-free technology and, as such, may become the gold standard for ovarian cancer diagnosis,” said Dr. Obermayr. “By combining the Parsortix technology with qPCR analysis, we achieved an unprecedented high detection rate of cancer, even in early-stage patients, when conventional diagnostic methods failed.”
DNA analysis can also be applied to the Parsortix harvest. One such technique is the PointMan technology from EKF Diagnostics. In controlled experiments, combining Parsortix with PointMan has enabled highly sensitive detection of mutant KRAS in tumor cells isolated from whole blood. Tumor cells with wild-type KRAS are more likely to respond to EGFR inhibitors such as cetuximab, and the ability to identify KRAS status in CTCs promises future prescribing of these drugs on the basis of liquid biopsy.
Together, these types of capabilities will enable identification of patient populations that will respond to molecularly targeted drugs. In the year that President Obama unveiled the Precision Medicine Initiative, the research and clinical community now has precision-guided munitions to add to their arsenal.