New Window into Brain Metastasis Using Modified CTC and cfDNA Technologies

Anthony M Magliocco MD

Many cancers metastasize to the central nervous system including the brain and its coverings, the leptomeninges. These cancers are difficult to treat and monitor. The so called “liquid biopsy” is making excellent progress in monitoring disease progression and response using blood samples in patients with disseminated solid tumors. In this technique, either circulating tumor cells (CTCs), or cell free DNA (cfDNA) can be harvested and captured for analysis.

These new “real time” monitoring methods create a new opportunity to monitor cancer progression and evolution while patients are actually under therapy- for example are the number of circulating tumor cells increasing, or is their phenotype evolving into something different.

For example, metastatic breast cancer cells may change their receptor status to go from estrogen receptor positive to negative, or they may acquire a new targetable change, for example acquiring HER2 over expression.

Images of circulating tumor cells captured in the Cell Search System

Intact tumor cells might also allow for examination of activation of signalling cascades and perhaps a pharmacodynamic read out.  One of the challenges of CTCs is their extreme fragility- with current method they generally need to be captured and examined within 48-72 hours. These cells also tend to be exquisitely rare, especially in early stage disease raising questions as to how representative they may be of the systemic disease.

 

Cell Free DNA cfDNA

A complementary assay method,  the measurement of cell free DNA (or cfDNA), provides a new application for monitoring cfDNA in patients. It has found widespread use monitoring lung cancer patients for the development of tyrosine kinase resistance while under therapy.  A common mechanism of resistance is the the switch or evolution to EGFR T790M mutation that portends the looming end of response to first generation TKI drugs and an opportunity to intervene with a switch to the 3rd generation Osimertinib therapy.

Liquid Biopsy is a powerful method to enable real time monitoring of solid tumor evolution and response to therapy using a blood sample to cature CTCs and cfDNA. Unfortunately, this does not typically work for CNS tumors due to the blood brain barrier

Unfortunately tumors occurring or metastasizing to the CNS are not easily followed by blood tests due to the presence of the blood brain barrier. However, these tumors are in contact with the cerebrospinal fluid, a specialized liquid that circulates around the brain cushioning it.

This fluid can be extracted in small amounts for analysis using a spinal tap, or in some cases of patients with malignancy an in-dwelling catheter is placed to enable CSF to be drained off to reduce the central nervous system pressure. This CSF fluid is traditionally sent to pathology for cytopathology analysis. Unfortunately standardized cytopathology methods do not lend themselves to evaluation of rare cells and molecular events in CSF.

 

Because CSF is very similar to blood, we reasoned that liquid biopsy methods used for blood samples might be potentially adapted for use on CSF samples. Recently, the Moffitt teams of the Neuro Oncology group, led by Dr Peter Forsyth and the Morsani Molecular Laboratory teamed up to attempt to modify the liquid biopsy procedures currently used for blood to adapt for CSF.

The current CTC platform in use at the Morsani Molecular Laboratory at Moffitt is the CellSearch system which is designed for magnetic capture of EPCAM expressing carcinoma cells in blood followed by evaluation of keratin expression in an automated scanning step with exclusion of non- specific cells using stains for nuclei and lymphocytes.

The adaptation of this system for analysis of melanoma in CSF was not trivial, as the volume of CSF is significantly less requiring adjustments on the liquid handling approaches. Further, the Neuroncology team was particularly interested in metastatic and primary melanomas of the central nervous system. This required changing capture antibodies to CD146 which targets melanoma and visualization with MelPE.

The Moffitt Morsani Molecular Laboratory has Developed New Methods to Monitor Melanoma in the CSF using both CTCs and cfDNA approaches

We also determined that DNA could be extracted and sequenced from CSF using both the NGS sequencing methods and MassArray systems. Further, we determined that melanoma cells captured from CSF can be grown ex-vivo and cultured for further analysis.

These advances create new opportunities to apply advances from personalized oncology to patients with metastatic melanoma in the CSF and central nervous system enabling potential real time adaptation of treatment strategies based on directly monitoring tumor molecular responsiveness to therapy in real time \\.

REFERENCES

Fedorenko IV, Evernden B, Kenchappa RS, et al. A rare case of leptomeningeal carcinomatosis in a patient with uveal melanoma: case report and review of literature. Melanoma research. 2016;26(5):481-486. doi:10.1097/CMR.0000000000000274.

Neuro-Oncology, Volume 19, Issue suppl_6, 6 November 2017, Pages vi46,https://doi.org/10.1093/neuonc/nox168.183 Published: 06 November 2017

 

Leave a Reply

Your email address will not be published. Required fields are marked *