Imaging Solutions for Cancer
Sampling circulating tumor cells (CTCs) in blood is a relatively noninvasive method for assessing tumor status or response to treatment. But CTCs present a number of difficulties for practical clinical use. Historically, these have been detected by antibody capture and cytokeratin staining. However, in 40–60% of cases no CTCs are recovered, even though they are likely present. These hurdles have hindered clinical use of CTCs as biomarkers.
Biocept says that OncoCEE™, its clinical biomarker platform, can capture rare CTCs with near 100% efficiency. The technology uses microfluidic channels to flow sample over a streptavidin-coated chip. The entire unit sits on a slide surface.
A cocktail of capture antibodies boosts recovery rates compared to other methods that use a single capture antibody, maintains a company official. Unwanted, non-antibody-bound cells flush through the system, keeping background levels low.
“What’s unique about our device is that there is a glass cover slip that allows users to take the entire unit, right after CTC capture and staining, to a microscope for direct manual analysis, where the person looking through the eyepiece can identify cells based on antibody stain and perform CTC enumeration,” said Farideh Bischoff, senior director, translational research and CLIA development at Biocept.
In early clinical testing, the OncoCEE was comparable to the Veridex (a J&J subsidiary) platform in its ability to enumerate CTCs from blood, according to Bischoff. It also offers an additional advantage of being able to conduct further testing on captured cells directly within channels, such as FISH for aneuploidy detection or gene-amplification studies that could be used to look for genetic biomarkers like Her2, Bischoff noted.
Harold Garner, Ph.D., executive director of the Virginia Bioinformatics Institute at Virginia Tech, introduced the hyperspectral imaging microscope, a new technology developed at the university. It enables researchers to scan each pixel of a microscopic image from 400 to 800 nanometers, quantifying as many as 15 fluorescent biomarker peaks in each scan. A single cell contains hundreds of pixels, so this type of imaging creates a very dense “cube” of data for every cell, permitting a view deep into subcellular compartments for markers of interest, he explained.
Dr. Garner’s team has put together a set of 10 markers for breast cancer and is currently optimizing similar sets for lung and colon cancer. Using data from breast cancer touch preps (where cells are collected from a tumor cell by touching a slide to it), Dr. Garner has shown that hyperspectral imaging microscopy correlates closely to standard 0 to +3 pathology scoring in treatment decision-making, but with a much higher density of information. The data is then entered into an interactive database where the user can view the contributions of each of the biomarkers across an entire image and in individual cells and tissues.
The hyperspectral imaging method addresses the problem of variability between labs in scoring of tumor cells, Dr. Garner pointed out.
“It’s very difficult to compare results from one laboratory to another. By standardizing these hybridization cocktails and the instrumentation and the database and analytical technique, we address those issues so people can use data from all over and be confident that it’s comparable,” Garner said, adding that, at the same time, it provides extremely dense biomarker data that can be used for personalized cancer therapy.
NextGen Sciences has developed a biomarker workflow using multiple reaction monitoring (MRM), a mass-spec method that uses peptides and their fragments to identify and quantitate proteins.
In the first phase, a hybrid LTQ-Orbitrap analyzes biological samples to generate a list of hundreds of potential biomarkers over a period of about six weeks. The next step is to develop assays that can multiplex up to 30 proteins at a time in order to verify the putative markers.
Because the process is iterative and proteins “fall out” of the running after testing samples, all of the proteins in the initial set can be screened, said Michael Pisano, president and CEO of NextGen. The results at this point are relative; in the next phase of the workflow the assay is converted to an absolute assay by spiking in isotope-labeled peptides for quantification.
The final phase is validation, which can comprise several levels depending on the intended use of the assay. The highest validation standards are for GLP and GCP applications that are going to be used in a clinical setting.
Although MRM has been in use for quite some time, the application of the technique to proteins as opposed to small molecules presents some new challenges, explained Pisano.
“A lot of the things you do with small molecule work do not apply to biomolecules. For example when monitoring for levels of drug or metabolites, there is no endogenous level in the patient. We always have to consider that there are components that are potentially present in the patient samples,” Pisano concluded.