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Nov 1, 2013 (Vol. 33, No. 19)

Growing Clinical Biomarker Assays

  • Another important factor is assay development. “From a regulatory perspective, scientists must develop an assay that the FDA will approve and thus must know the newest regulatory guidelines. If assays have poor design style or manufacturing standards, it will be hard to get platform approval.”

    Throughput is an additional consideration. “This really depends on the patient population. Often therapeutics and companion diagnostics may be for specially targeted patient populations, so throughput here is not critical. But if you have 100,000 patients and 10 sites that are involved, then you must consider the optimal platform for this level of throughput.”

    But even the best platform requires something else—accessibility. “You need to select a platform that has unlimited access. New technologies such as NGS can be very useful, but at the moment, NGS is not widely available. While today it is largely used in research, in the near future, NGS will become much more user-friendly and commonly employed.”

  • Which Assay to Use?

    Click Image To Enlarge +
    Some companion diagnostic assays make use of fluorescent in situ hybridization (FISH), as does Dako’s automated IQFISH system. The system depicted here shows an IQFISH slide beneath the lens of a fluorescent microscope, and fluor-escently labeled cells appear on a screen.

    Almost every new oncology drug being developed today is targeted toward a specific protein or family of proteins. Thus, companion diagnostic assays are needed to help identify those patients who will benefit from a new drug. There are a number of methods and platforms for which the companion diagnostics can be applied. Dako, an Agilent Technologies company, focuses on immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and bright-field chromogenic in situ hybridization (CISH) assays on fully automated systems.

    According to Hans Christian Pedersen, principal scientist at Dako, “A companion diagnostic needs to be available wherever the drug is intended to be used. Thus, the selected technology must be adaptable into routine clinical practice and the costs secured through adequate reimbursement.”

    “Additionally, a companion diagnostic test must have a reasonable turnaround time, typically within one day, to avoid delays in providing test results to the oncologists making the treatment decision. IHC and Dako’s recently introduced IQFISH technology fulfill all of these criteria and also have the advantage that most pathology laboratories are performing these tests today, and therefore have the infrastructure, know-how, and skills necessary to perform these assays.”

    Pedersen adds that since most new drugs are directed at a protein, and the protein represents the functional entity in the cell, it is important to look at the protein in the context of both cellular localization (membrane/nuclear/cytoplasmic) and being able to assess protein expression levels directly in the context of the tissue. “Again, IHC allows for this type of determination and performs very well,” Pedersen notes.

    For the future, Pedersen agrees that NGS and quantitative polymerase chain reaction (PCR) may be used more in the clinical setting. “We are already seeing companion diagnostic collaborations that utilize quantitative PCR technology. At the moment, these technologies represent big potential, but also big challenges.”

    “With the rapid pace at which the technology is moving, today’s NGS platform may differ from tomorrow’s NGS platform. It is uncertain what effect NGS will have on the quantitative PCR-type tests being developed today. However, it is crucial for pharmaceutical companies to select a technology platform for their companion diagnostic that won’t be outdated by the time the drug is launched.”

  • Epigenetic Markers Found for Head and Neck Cancers

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    Epigenetic markers for head and neck squamous cell carcinomas have been identified that could be clinically exploited as biomarkers for early precancer screening. [Eraxion/iStock]

    Citing the potential for noninvasive tests capable of the early detection of head and neck cancer, researchers based at Queen Mary University in London announce that they have identified epigenetic changes in cancerous cells that are not seen in healthy cells. In particular, two genes (GLT8D1 and C6orf136) were found to be differentially expressed in head and neck squamous cell carcinomas (HNSCCs). Using methylation-specific quantitative PCR, the researchers confirmed that the promoters of GLT8D1 and C6orf136 were hypo- and hypermethylated, respectively, in HNSCC tissues.

    The researchers published their results October 1 in the journal Cancer, in a paper entitled “Identification of FOXM1-induced epigenetic markers for head and neck squamous cell carcinomas.” In this paper, the researchers describe how they analyzed clinical specimens of malignant tissue from 93 cancer patients from Norway and the United Kingdom. These specimens were compared with either tissue donated by health individuals undergoing wisdom tooth extractions, or with noncancerous tissue from the same patients.

    In their study, the researchers focused on FOXM1, an oncogene that has been implicated in all major forms of human cancer. They hoped to identify the earliest cancer-predictive epigenetic biomarkers accrued during aberrant cell proliferation induced by FOXM1. They identified a panel of FOXM1-induced differentially methylated genes, including C6orf136, MGAT1, NDUFA10, and PAFAH1B3, which were hypermethylated, and SPCS1, FLNA, CHPF, and GLT8D1, which were hypomethylated.

    In addition, the researchers further investigated and validated the expression profiles of these candidate genes in head and neck tissue specimens. The researchers noted that they measured differential promoter methylation and gene expression in clinical specimens using commercially available bisulfite conversion kits and absolute quantitative PCR, respectively.

    Of the four hypomethylated genes, three genes—FLNA (filamin A, alpha), CHPF (chondroitin polymerizing factor), and GLT8D1 (glycosyltransferase 8 domain containing 1)—were found to be consistently upregulated, although only GLT8D1 was statistically significant in both HNSCC cohorts. The researchers asserted that their promoter methylation analyses confirmed that the promoters of these three genes were indeed hypomethylated, a result that the researchers say suggests that promoter demethylation may be a mechanism responsible for gene expression hyperactivation: “Given that the expression of the three genes was aberrantly upregulated in both cohorts of HNSCC tissues, we speculate that they may play a role in oncogenesis.”

    Of the four hypermethylated genes, only one gene, C6orf136, was found to be consistently downregulated in both HNSCC patient cohorts. Their methylation study, the researchers said, confirmed that the promoter of C6orf136 was hypermethylated in HNSCC, suggesting that downregulation of its gene expression may be a result of promoter hypermethylation.

    Lead researcher Muy Teck-Teh, M.D., from the Institute of Dentistry at Queen Mary, said: “In this study we have identified genes which were either over- or underexpressed in head and neck cancer. The expression of these genes was inversely correlated with particular DNA methylation marks, suggesting the genes are epigenetically modified in these cancers.”

    Dr. Tuck-Teh said that these epigenetic markers could be clinically exploited as biomarkers for early precancer screening of head and neck cancer, but he also noted that further work would be needed, as his group’s work is still in the discovery stage. At present, there is still no diagnostic test.

    “The eventual aim would be to test asymptomatic patients and/or people with unknown mouth lesions,” added Dr. Tuck-Teh. “An advantage of epigenetic DNA markers is that it may be possible to measure them using noninvasive specimens. So it could enable the use of saliva, buccal scrapes, or blood serum for early cancer screening, diagnosis, and prognosis.”

    Last year, in the International Journal of Cancer, researchers from Queen Mary University published a paper in which they described a genetic test that they had developed for the early detection of oral cancer. That test, called the quantitative Malignancy Index Diagnostic System, quantifies the expression levels of 14 genes associated with the FOXM1 cancer gene, and converts the measurements into a diagnostic score that indicates the risk of a lesion becoming cancerous.

    This work, said the researchers, showed that aberrant upregulation of FOXM1 “brainwashed” normal cells by reprogramming the methylome, changing its landscape toward those found in cancer.

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