A Tale of 2 Biomarkers: CTCs and cfDNA are Key to Managing the Plethora of New Trial Options

By Anthony M Magliocco MD


We are currently living in revolutionary times when it comes to cancer therapy and treatment options. There are literally hundreds of clinical trials under way evaluating dozens of new therapeutic compounds and combinations of therapy. In fact there are so many trials that its difficult to always find enough patients for them and also to design them to produce the level of evidence expected in traditional multi-armed phase III trials.

There are hundreds of open clinical trials testing dozens of compounds and combinations of therapy


Indeed, we seem to be arriving at the point where cancer therapy really is being tailored and delivered to the individual. This shift from evidence based conventional cancer therapy clinical trials, to newer, matched and “N of One” trials creates new challenges for the oncologist and diagnostic laboratory industry.

In a traditional trial, selection and enrollment would depend on results of a key biomarker such as HER2 overexpression for Trastuzumab therapy

Traditionally a biomarker would be required to select a patient for a specific therapy. For example, in breast cancer, presence of HER2 over expression or amplification was a marker to help select patients for enrollment in a trial. Because the frequency of the biomarker was relatively common in a common disease, it was possible to build a well powered phase III trial to collect convincing evidence that on a population based setting that this was an effective strategy. This led to FDA approval and the development of companion diagnostics such as the Herceptin Test.

Fast forward twenty years or so. Now we have so called “basket trials” where cancers can be tested with complex genomic tests that will evaluate hundreds of genes, for example the Moffitt STAR assay that covers 170 actionable mutations and alterations resulting in detection of relatively rare, but still actionable mutations in many tumors. However the mutations are variable and the choices for treatment complex meaning that more than one combination of therapy could be considered. Whats an oncologist to do?

Going forward, it appears that if patients cannot be managed or enrolled in large trials, there will need to be an approach to effectively manage the so called “N of One” patient. In fact, it could be considered that almost every patient is a now unique in some way and could be classified as a rare disease

With a movement away from classical evidence based clinical trials toward n of one trials, off label therapy, and basket trials new approaches to companion diagnostics are urgently needed

In this situation, it appears that physicians will need to act on “best available evidence” or actual bioinformatics or other predictive models of possible response based on understanding the underlying molecular circuitry in the cancer in question. In this situation a “best guess” is made for assignment of therapy (either in a basket trial or in an off-label situation).

This approach can be problematic and has numerous complications such as the possibility of providing futile or toxic treatment. Fortunately, there are plenty of new advances in technology that might address this problem. The most help may come from the so-called “liquid biopsy”. Which is essentially usually a simple blood test evaluated with a exotic new technology.

Liquid Biopsy

The main components of a liquid Biopsy include circulating normal cells (WBC, Platelets, RBCs) possibly circulating living cancer cells (CTCs) and bits of dying cancer cells (cell free DNA, miRNA etc).

A Story of Two Biomarkers CTCs and cfDNA

CTCs Circulating Tumor Cells

The CTCs are very fragile, rare and hard to detect but give a window into the living cancer in the patient as treatment progresses. These cells can be further evaluated to determine if they are proliferating or if certain signalling pathways are active. In fact they can also be harvested, sequenced, and in some cases grown and expanded in culture.

Cell Free DNA cfDNA

Cell Free DNA or cfDNA gives a more stable read out of the tumor load, and mutation composition of the DNA, or at least the DNA leaking into the blood. It might be expected that when a new treatment begins, cancer cells in the host may undergo death and leak DNA into the blood. Consequently there could be an initial “Spike” in the amount of circulating DNA – this could be a positve signal. In other instances, cfDNA might indicate if there is residual cancer left in a patient after a surgery was completed that was initially intended to remove all disease giving a type of molecular staging. If a therapy is working well we would expect that CTCs and cfDNA would decrease and perhaps become immeasurable. On development of resistance there would be detection of new clones and expansion of the concentration of CTCs and cfDNA fragments.

Liquid biopsy provides a means to monitor tumor response to therapy in a dynamic and real time manner giving unprecedented opportunities to modulate treatment and truly personalize therapy for cancer patients

I expect that the twin technologies of CTC and cfDNA analysis will become more valued by oncologists, patients and payers as these tools will provide a way to dynamically monitor tumor response to treatment and provide immediate evidence of efficacy of a therapy or also of impending relapse potentially allowing a window of opportunity to adjust treatment.


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.