American Association for Clinical Chemistry
Better health through laboratory medicine
September 28, 2006

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In this Issue...

Detecting Early Breast Cancer
Study of Ultrasensitive Immunoassay Method Shows Promise
by Julie McDowell


Breast cancer is currently diagnosed by triple testing: clinical examinations; screening using imaging techniques including mammography and ultrasonography; and biopsies. Early diagnosis is important in treating and managing this disease, and one of the primary reasons that breast cancer mortality rates have declined in recent years. One of the key tenets in advancing early diagnosis is developing more sensitive and specific in vitro diagnostic testing techniques. This issue of Strategies looks at early research on developing ultrasensitive immunoassays using multiphoton-detection techniques that could eventually make laboratory testing an integral part of early disease detection.

While breast cancer is the most common cancer afflicting women, research has found that the five-year survival rate can be as high as 97% if the cancer is small, of low grade, and hasn't spread to the lymph nodes. Therefore, clinicians want to detect the breast cancer at the earliest stages, which isn't always possible with imaging technology alone, explained John Tomaszewski, MD, Professor of Pathology at the University of Pennsylvania in Philadelphia .

“If you look at the positive predictive value of those kinds of tests for breast cancer, it's not real good. It's on the order of 20—25%, maybe,” he said. “Part of it has to do with who you are screening and part of it has to do with the sensitivity and specificity of the radiological techniques.” Clinicians typically see a lot of biopsies for benign disease, and would like to have a simpler, less expensive screening tool that incorporates an in vitro testing technique with better sensitivity and specificity. The best situation would bring together both imaging and in vitro testing techniques. “There's a need for biomarkers in blood to enhance the efficiency of screening techniques,” Tomaszewski added. “To date, in breast cancer screening, there really hasn't been anything that's been effective.”

Towards Ultrasensitive Detection

Biomarkers for early detection continue to be a major focus of cancer research. At a recent meeting sponsored by the American Association for Cancer Research (AACR), findings of numerous studies on potential biomarkers for prostate, colorectal, and lung cancers were presented. The studies included analysis of two genes—REG1A and EXTL3—that researchers found were overexpressed in colorectal cancer, while another study looked at how levels of thymidylate synthase (TS) may help predict survival after colorectal cancer. There were also findings presented on the role of the human aspartyl bata-hydroxylase or HAAH in predicting prostate cancer. Additional information on findings from this meeting, “Molecular Diagnostics in Cancer Therapeutic Development” held this month in Chicago, can be found on AACR's Web site, www.aacr.org.

But an important component of researching potential biomarker panels for breast cancer is developing an ultrasensitive assay. One of Tomaszewski's collaborators, Andrzej Drukier, MD, Chief Executive Officer and Chief Science Officer, BioTraces, Inc. (Herndon, Va.), has confronted the problem of assay sensitivity by developing a new immunoassay using multiphoton detection (IA/MPD) and derived supersensitive Super ELISA assays, which have been shown to increase sensitivity 200 to 1000 fold. Drukier, who has patented this technology, is basically using this technique to subtract noise from the signal—which results in a better signal, explained Tomazewski.

Drukier and Tomazewski's clinical research group, which includes scientists from the University College London's Department of Surgery and the Pittsburgh Cancer Institute, recently published results of a study using IA/MPD technology to analyze data sets, including sets of imaging data and serum samples from women with breast cancer who were not receiving treatment, and sets of serum samples from healthy women. In total, the researchers looked at 250 patient samples and 95 controls, and achieved 95% specificity and sensitivity. Their study appeared in the September issue of the Journal of Proteome Research (2006, 5, 1906—1915; http://pubs3.acs.org/acs/journals/hot_article.page?in_manuscript_number=pr0600834).

“This technique uses a sensitive binding assay that allows us to look at low frequency, or low concentration proteins in the serum,” explained Tomaszewski. “We looked at a number of different proteins in the serum, including PSA, interleukins, and vascular endothelial growth factor, or VEGF. At the level of sensitivity of our assay, we could measure all of these proteins in all patients. If you do a traditional immunoassay, you might only measure detectable levels in a third of patients, so you have a lot of non-informative patients. But in our assay, we get information from essentially all the patients.”

Looking Ahead to Non-Linear Areas

Tomaszewski emphasized that this research is in the development stages. There's a lot of research, as well as regulatory hurdles to clear, before this testing technology, which is currently a manual assay, comes to the lab. The research group is currently looking at other patient cohorts, but the results are promising in that there appears to be good separation between malignant and benign lesions using this testing technology.

Nevertheless, these findings indicate the direction that clinical laboratories will be going in the future, said Tomaszewski. For screening purposes, this technology could identify patients with a higher risk panel. Patients would have their blood tested during their annual physical, and if there were low levels of biomarkers such as interleukin, VEGF, and TNFα, then they might be classified as low risk, and told to continue having annual examinations. But if there were high levels, these patients could be characterized as high risk, and clinicians might want to supplement the annual exam with intensive imaging techniques.

“There are other ways to do this and there will be competing technologies out there. For example, mass spectrometry technologies could be used and there are other molecular-based sensitive assays,” he said. “The fundamental concept is that laboratories will be measuring larger numbers of proteins using assays which have broader ranges with linear dynamics, and this will provide us with more sensitive assays. Laboratorians have to have methods with larger linear operating ranges if they are going to be able to measure and clinically validate some of these new tests for cancer.”