Multiplexing in Cancer Diagnostics

Vol. 14 •Issue 10 • Page 72
Multiplexing in Cancer Diagnostics

Scientists say multiplexing provides a new life for biomarkers.

Tumor-specific and tumor-associated biomarkers have been of growing interest for diagnostics for more than 20 years. Traditionally, single protein immunoassays have been used to evaluate recurrence after therapy or predict cancer risk in conjunction with other symptoms. However, sensitivities of these assays for diagnostic screening have been limited since complex diseases such as cancer cannot readily be linked to changes in levels of single proteins. The quest for earlier diagnosis of cancer prior to visible symptoms calls for novel diagnostic tools.

Following the footsteps of the genomics revolution, rapid evolvement in the field of proteomics in recent years resulted in a new focus on proteins as diagnostic tools in cancer. Proteomics strive to analyze all proteins of a cell or sample using various techniques, in particular mass spectrometry and 2Dgel electrophoresis. The results of these technologies are aligned against databases to identify and categorize proteins or protein families. The wealth of data from proteomics poses a number of basic challenges to the diagnostic field:

1. Which peaks/proteins are meaningful biomarkers?

2. Which biomarkers can be combined to achieve additive diagnostic value?

3. How can multiple markers be tested efficiently?

4. How should the data be analyzed to result in a reliable and reproducible clinical diagnosis?

The translation of knowledge into clinical use will require a multidisciplinary approach from basic researchers, statisticians and clinicians to integrate diverse technologies for the patient’s benefit.

Beyond Selection of “The One”

Although general proteomic patterns in patient samples have been tested for their diagnostic value, the recent consensus places proteomics into biomarker discovery that needs to be followed by a selection of a limited set of markers for development into diagnostic kits. The ultimate test objective and requirements for sample material (i.e., cells, serum, urine, etc.) can be used as a starting point to direct the researcher through the results from proteomics screening and help with the selection of biomarkers for further development.

By definition, biomarkers ideally should be specific for certain tumors or cancerous conditions. Markers that only indicate the presence of a tumor in general need to be combined with tissue specific analyte(s). If molecular tests are out of question, for example due to cost or technology prohibitions, the markers of choice have to be present in sufficient abundance in the sample to be detectable. Also, serum or urine soluble markers are preferable to ease sample collection; marker stability post sample collection is beneficial.

Once certain markers of interest have been identified, specific antibodies for detection need to be generated. Two different antibodies are preferable to increase assay specificity. Latest at this point, some preliminary thought should be spent on potential patent and licensing issues.

The combination of more than one marker to detect specific cancers is a science by itself and involves the application of advanced statistical models. Sensitivity of several markers should be additive and also result in increased specificity for the condition of interest. For example, pre- and post-menoposal or age-related markers could be combined to increase sensitivity in the general patient population. The combination of several markers can greatly increase sensitivity compared to single protein screens.

Basics of Multiplexing

The analysis of several biomarkers for cancer diagnostics quickly reaches limits in clinical laboratory practice. Carrying out, for example, four or more individual tests on each patient sample is tedious on a large scale, requires large amounts of sample material and introduces additional potential for errors. This bottleneck could be addressed by multiplex assays—the combination of several separate biomarker tests in a single tube.


A number of technologies are available for multiplex assay development. Established ELISAs are coming of age as the limited number of enzyme labels reduces multiplexing capabilities. Fluorescent immunoassays (FIAs) are the closest alternative replacing the enzyme label with differently colored fluorescent tags specific for each marker. Improvements such as nanocrystals and time-resolved fluorescence push FIA sensitivities into the range of standard ELISAs.

Bead-based sandwich assays carry the principle of FIAs to the next level. The primary antibody is immobilized on a fluorescent bead, which comes in up to 100 different colors. Detection is accomplished using a secondary antibody linked to a fluorescent tag. Each bead is analyzed individually in a flow-through fashion and the combination of the two fluorescent signals indicates binding of the analyte to both antibodies.

Protein arrays immobilize capture antibodies in a distinct micro-spot pattern separately from each other on a glass slide or on the bottom of a microwell plate. Up to 13 analytes can be tested in a single well. Sample is added to bind to the different antibodies followed by a mix of secondary antibodies that are fluorescently tagged. Special array readers detect the fluorescent signal on the individual spots. While the preparation of the initial array can be challenging, the final assay in the clinical laboratory can be kept simple with protocols that resemble standard ELISA procedures. In addition, spatial separation of captured analytes allows the use of only a single-color fluorescent tag, thus simplifying assay development.

The final platform choice will depend on the applicability at the end-user.

Data Analysis

Once the different assays have been combined into a multiplex format, the question arises how to separate the data at the end. In the simplest scenario, only one single detection label would be used and the mean signal from all markers recorded. This method, however, sacrifices important information on which specific marker is up- and which ones are down-regulated. More sophisticated data analysis looks at each marker individually and subjects all data to an algorithm, which could result in a percent risk of having cancer. High-risk patients could then be referred for biopsy tests.

Again, a look at the details quickly reveals potential problems. For example, if one or a few markers are up, and a few others are down, what does this mean for diagnosing cancer? The cut-off levels of single marker assays also need an overhaul in the light of multiplex analysis. Each biomarker adds another dimension of complexity to the data analysis while at the same time the sight should be kept on user friendliness of the final result. Biostatisticians are developing novel algorithms to analyze data for multiplex diagnostics.

Money Talks

When the technical issues have been solved and a powerful multiplex assay arrives in the clinic, it is time to talk about money. When performing several individual assays, each is priced separately. Development costs for running a multiplex assay, however, are more than additive for each analyte added into the picture and a diagnostics company will have to adjust pricing accordingly. On the other side, multiplex assays will probably not be much more labor intensive than single-analyte assays in the clinical laboratory. Many biomarker assays already are being reimbursed by “gap-filling” or “cross-walking” to codes from 1984. The cost associated with the proteomics revolution and multiplex diagnostics will need to be reflected in insurer’s reimbursement levels.

Dr. Verch is research scientist, Research and Development, Fujirebio Diagnostics Inc. Elizabeth Somers is director, Business Development, Fujirebio Diagnostics Inc.

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