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

 

FDA approves osimertinib for first line use in lung cancer with EGFR mutation

By Anthony Magliocco MD

TAGRISSO delivered unprecedented median progression-free survival of 18.9 months versus 10.2 months for EGFR-TKIs (erlotinib or gefitinib) in 1st-line EGFRm NSCLC

 

the US Food and Drug Administration (FDA) has approved TAGRISSO® (osimertinib) for the 1st-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) mutations (exon 19 deletions or exon 21 L858R mutations), as detected by an FDA-approved test. The approval is based on results from the Phase III FLAURA trial, which were presented at the European Society of Medical Oncology 2017 Congress and published in the New England Journal of Medicine.

 

The results of the phase III FLAURA trial were impressive with dramatic improvements to progression free survival.

The FLAURA trial compared TAGRISSO to current 1st-line EGFR tyrosine kinase inhibitors (TKIs), erlotinib or gefitinib, in previously untreated patients with locally advanced or metastatic EGFR-mutated (EGFRm) NSCLC. TAGRISSO met the primary endpoint of progression-free survival. PFS results with TAGRISSO were consistent across all pre-specified patient subgroups, including in patients with or without central nervous system (CNS) metastases. Overall survival data were not mature at the time of the final PFS analysis.

 

Osimertinib was previously approved for use as second line therapy in patients who had progressed on a TKI treatment or for those whose tumors developed a T790M mutation confering resistance to the first generation TKI therapies.

Osimerinibs mechanism of action is thought to be irreversible binding to the EGFR receptor.  Perhaps this explains the improvement in PFS compared to other TKIs. These findings are potentially practice changing.

Despite these impressive improvements in PFS almost all patients eventually fail targeted therapies with TKI agents. Consequently more work is needed to understand the biological mechanisms of resistance and progression in lung cancer patients to enable more effective therapies to be developed.

https://www.onclive.com/web-exclusives/fda-approves-frontline-osimertinib-for-nsclc

Bringing Digital Droplet PCR into the Cancer Clinic for Treating Progressive Lung Cancer

By Anthony M Magliocco MD

Digital Droplet PCR, or ddPCR is a phenomenally sensitive, specific, and most importantly, precise method to measure target DNA.

One important application for this technology that is now reaching the clinical laboratory is the development of tests for monitoring circulating cell free DNA that is released from cancer cells.

The challenge of detecting cfDNA is monumental.  A tube of blood is loaded with a rich variety of complex biomolecules and cells including lymphocytes, neutrophils, platelets, and of course red corpuscles.  These cells, including RBCs, are loaded with nucleic acids and whats worse, they can easily rupture during blood draws or pre analytical handling.

Advanced lung adenocarcinoma, (NSCLC) is probably the single tumor type that has caused the largest advances in personalized oncology of solid tumors. Investigations into the molecular biology of this disease has uncovered the fact that NSCLC is a heterogeneous disease defined by distinct molecular subtypes and clear molecular driving pathways. Further the discovery of targetable driver mutations including presence of EGFR mutations among others has led to pivotal clinical trials proving the efficacy of the Tyrosine Kinase inhibitor Drugs (TKIs).  Further it seems that lung tumors with EGFR mutations are more likely to arise in women and non-smokers.

Despite the remarkable efficacy of TKI therapy in many patients with advanced NSCLC, the disease is relentless and frequently overcomes treatment by developing resistance mechanisms. The commonest mechanism of resistance is acquisition of EGFR T790M mutation which further enhances the activity of the EGFR pathway, driving growth of the lung cancer cells.

 

 

Fortunately, third generation non-competitive inhibitors of EGFR were developed that can overcome the development of T790M resistance -osimertinib or Tagresso from AZD.

When Osimertinib first became available it was necessary to develop an ultra sensitive assay to detect the mutation in tissues and blood.

The Moffitt Cancer Center Morsani Laboratories, when faced with this challenge, decided to use a digital PCR approach because of its superior sensitivity, specificity and reasonable cost of operation.

Dr John Puskas worked diligently to develop and validate a cfDNA assay for EGFR T790M mutation using ddPCR for CLIA at the Moffitt Cancer Center.  Different PCR platforms were evaluated but the Bio-Rad QX200 was eventually selected for use.

The assay was superbly sensitive and precise as well as convenient to run.  The assay can detect mutant EGFR T790M down to 0.1% and can easily monitor blood concentration over time to give assessment of disease progression

 

 

The development of accurate and specific cfDNA assays to precisely monitor cancer progression in the metastatic setting is an important tool to potentially enable oncologists the opportunity to determine more rapidly f tumors are responding to treatment or not and make adjustments.

This novel opportunity to trace tumor evolution in real time, at a molecular level, could play a role in trials and treatments of the future where treatments are carefully adjusted to avoid the inadvertent development of treatment resistance due to over treatment.