Pictures of the Future      Fall 2008

Answers in the Blood

Accurately diagnosing illnesses such as cancer can be an extremely complex and protracted process. Yet there are now many tests that provide a fast and foolproof identification of diseases in the lab—often using just a few drops of blood.

As he was being rushed into the emergency room he was sure he would pass out. Gasping for breath and quite obviously in the grip of acute nausea, the only way he could fight off the overwhelming feeling of suffocation was by maintaining an upright position. Seeing what appeared to be all the symptoms of a heart attack, doctors set to work immediately. Yet after a thorough examination, including an ECG, they still could not be 100 % sure of their diagnosis. They therefore resolved to monitor the patient’s condition with the help of a new and extremely precise test procedure. This proved to be exactly the right decision, since over the next few hours one of the patient’s blood values continued to rise, as doctors suspected it would.

Later, an examination with a cardiac catheter confirmed their suspicions with the discovery of a stenosis in one of the patient’s cardiac arteries—the cause of the heart attack. Immediately thereafter, the patient’s cardiologist opted to insert a stent—a small metal tube to dilate the artery in question.

Released in 2006, Troponin-I-Ultra is the name of a test developed by Siemens Healthcare Diagnostics at its U.S. research labs. The test is used in conjunction with the ADVIA Centaur, a fully automatic immunoassay system from Siemens, and serves to detect the presence of the protein troponin in blood with the aid of receptor molecules (see graphic).Troponin, which normally occurs only within the heart, is released into the blood as a result of the death of cells (necrosis) in the cardiac muscle (miocardium). In other words, its presence provides an extremely specific indication of damage to the cardiac muscle. Increased troponin levels in blood serum are detectable around three hours after the beginning of a heart attack, rising to a maximum within around 20 hours after the attack, and returning to normal after two to three weeks.

Compared to the Siemens test, no other procedure is quite so precise nor able to deliver such an unequivocal indication of an increase in troponin I levels. The Troponin-I-Ultra test in conjunction with the ADVIA Centaur was the first fully automatic analytic method to meet current guidelines of expert groups at the American College of Cardiology Committee in Washington, D.C. and at the European Society of Cardiology.

Thanks to its sensitivity, the new test obviates the need for multiple exams, which are often required in the case of imprecise results. Obviously, that saves time and money. According to Dr. Till Neumann, Senior Physician at the Cardiology Clinic of the University Hospital in Essen, Germany, the introduction of new and more sensitive troponin tests will mean fewer borderline results—and therefore a greater significance for troponin-based diagnosis in everyday clinical practice.

Troponin-I-Ultra is a prime example of the successful development of a range of new biomarkers that  will enable earlier or more precise lab diagnoses of a variety of medical conditions. All that is required for the test is a sample of the patient’s blood. This is much easier, quicker and less expensive than a scan. Test developers are therefore working to make Troponin-I-Ultra—which is particularly applicable to management of patients suspected of experiencing a heart attack—even more precise and meaningful.

Hormones from the Heart. The ultimate goal, however, is to be able to recognize life-threatening conditions long before they become critical. For instance, another test—one that detects the presence of B-type natriuretic peptide (BNP), a biomarker that is indicative of cardiovascular disease, can also be employed to help doctors reach a quick and accurate diagnosis. BNP is a hormone that is formed and secreted in the left or right ventricle of the heart during cardiac insufficiency, otherwise known as heart failure.

BNP tests have been in use for a number of years now and, according to Alan Burkhardt, who develops such procedures for Siemens Healthcare in Tarrytown, New York, "they are very effective in detecting heart conditions in the absence of any other clear symptoms." The prognostic power of the BNP test has also been corroborated by the Heinz-Nixdorf Recall Study on the Early Diagnosis of Cardiac Disease, which was conducted at the University Hospital in Essen, Germany.

This study involved the examination of almost 5,000 men and women from the Ruhr area of Germany in order to determine their risk of suffering from a cardiovascular complaint at some time in the future. The study revealed that today’s threshold value of 100 pg/ml —1 pg is a trillionth of a gram—should be reconsidered, and that in the future threshold values specific to age and gender should be assigned with a view to facilitating the diagnosis of myocardial damage at a very early stage.

"The presence of troponin and BNP concentrations are important diagnostic indicators in today’s cardiology armamentarium. With the Troponin-I-Ultra and the B-type natriuretic peptide tests, we have two procedures that are of use both in emergency situations and in the early detection of cardiovascular disease," says Burkhardt.

Blood Test instead of Liver Biopsy. The ELF™ (Enhanced Liver Fibrosis) test was likewise developed to provide an early diagnosis and monitoring tool for liver fibrosis. Liver fibrosis is the response of the liver to chronic injury by viruses, alcohol abuse or metabolic disease-related conditions. It is manifested in an accumulation of connective fibrotic material leading to a stiffening of the liver tissue.

Liver function is compromised if the condition becomes sufficiently advanced, and it can culminate in a life-threatening cirrhosis and complete liver failure. Liver fibrosis is also associated with a significantly increased risk of liver cancer. The gold standard today for diagnosis of liver fibrosis is the biopsy. Here, a doctor uses a needle-like instrument to remove a small sample of tissue from the liver, which is then examined under a microscope. Such a procedure is not without risk. It can lead to significant complications such as internal bleeding and is not well tolerated by patients. Moreover, a liver biopsy is also relatively imprecise, since there is a significant sample error and no guarantee that the needle will actually encounter abnormal fibrotic tissue. For these reasons, repeat biopsies for diagnosis or monitoring purposes are not desirable. The clinical community has therefore long hoped for the development of a  non-invasive liver fibrosis test for patients with chronic liver disease.

The new ELF test spares both the patient and physician the hazards of an invasive procedure. It measures three biomarkers in a simple blood sample, which when combined generate an Enhanced Liver Fibrosis score that is correlated to the level of fibrosis in the liver. The innovative feature of the new test is that it is the first blood test that measures direct markers of liver fibrosis that may accurately diagnose the presence of liver fibrosis in the first place, thus avoiding the problems and expense of unnecessary biopsies. The ELF score may provide physicians with a valuable new tool to better manage their patients with chronic liver disease.

The ELF score is being clinically validated for use in patient groups on its own and in combination with Siemens imaging modalities at a number of sites in Europe and the United States. It is also currently under development for use on the Siemens ADVIA Centaur Immunoassay System, which offers laboratory customers a broad range of routine immunoassays consolidated on a single platform.

"Our Enhanced Liver Fibrosis test meets a previously  unmet medical need and is proprietary to Siemens Healthcare Diagnostics. The next step is to integrate the ELF test in our ADVIA Centaur immunoassay system and introduce it to major markets as soon as possible. Its availability will provide laboratories with a unique opportunity to offer their customers our innovative ELF Score as a routine test," says Dr. Andrew Beard, Liver Fibrosis Senior Marketing Manager at Siemens Healthcare in Tarrytown.

The ELF Score was first clinically validated in 2004 in a collaboration between Bayer Healthcare Diagnostics and the European Liver Fibrosis Group led by Professor William Rosenberg from Southampton University in the UK. Bayer Healthcare Diagnostics has since been acquired by Siemens and in Europe a CE-marked ELF score is currently available for routine use and pharmaceutical testing from IQUR Ltd, UK.

Early Detection of Septicemia. Doctors are under constant pressure to come up with fast and accurate diagnoses, i.e. to determine on the basis of the patient’s medical record and current symptoms the precise nature of a medical complaint. Yet often the problem is that medical conditions creep up slowly and only fully manifest themselves when it is almost too late to correct them. An especially insidious example of this is septicemia, a form of blood poisoning in which bacteria and other pathogens multiply at an explosive rate. Patients weak from a serious operation are particularly at risk from these hordes of hostile microbes. According to a large-scale study conducted by the German Sepsis Society for the years 2003 to 2006, there are 154,000 new cases of septicemia in Germany every year and 60,000 fatalities. That makes septicemia one of the biggest risks and most common causes of death for patients in intensive care.

Siemens Healthcare has therefore developed a new procedure that—like the Troponin-I-Ultra test—is able to detect the presence of low concentrations of a specific biomarker in the blood. In this case, the test shows the presence of a substance known as LBP (lipopolysaccharide binding protein), the production of which is stimulated by the rapid buildup of bacteria.

Regular tests of LBP levels in a patient’s body can provide early warning of the continuing presence of a local infection that has the potential to spread to the entire body, thus giving doctors a chance to prescribe the requisite medication before an infection gets out of hand. Yet what makes sepsis particularly dangerous is that while the outbreak of pathogens is often initially limited to a local area, the condition can in some cases, after a week or so, flare up again and spread rapidly and systematically to the rest of the body. It is often this "second hit" that proves fatal (see graphic).

However, the LBP test provides early warning of a continuing local buildup of bacteria, which is responsible for the second hit.  Other tests, on the other hand, such as those based on detection of interleukin 6, an endogenous pro-inflammatory cytokine (messenger molecule), only respond once the infection starts to spread. "In other words, the LBP readings help doctors in intensive care to quickly make the right decision and thus save the lives of patients who would otherwise quite likely die from an infection that is identified too late or not at all," explains Dr. Peter Zwerenz, Marketing Manager at Siemens Healthcare Diagnostics in Eschborn, Germany.

The new LBP test runs on Siemens’ Immulite immunoassay system. The Immulite-System is already in widespread use, and is therefore available to the majority of intensive care units.

Another challenge facing doctors is the choice of the right therapy, since not every form of medication is equally suitable for each patient. Take Herceptin, for example, a drug used in the treatment of breast cancer, but which is only suitable for tumors that produce a certain type of receptor: the HER-2/neu (human epidermal growth factor receptor). Herceptin blocks this receptor and therefore the signaling pathway that controls tumor development, thus inhibiting the growth of cancer cells.

However, the HER-2/neu receptor is present only on the surface of a maximum of one-third of all breast-cancer tumors; only those patients who have it can be helped by Herceptin. With this in mind, Siemens developed a test that measures the level of HER-2/neu receptor circulating in the blood. It is available both as a manual test and as an automated test that can run on the Advia Centaur.

The test was developed primarily by Walter P. Carney, PhD, at Oncogene Science Diagnostics, in Cambridge, Massachusetts. The company was acquired first by Bayer in 1999 and then by Siemens in 2006. In other words, the Siemens portfolio of companies now includes a highly creative enterprise for the development of biomarker tests.

"The HER-2/neu-Test received FDA approval for monitoring patients with metastatic breast cancer in 2000," Carney explains. In Europe, where many clinics now use it on a routine basis, it has been on the market for several years. "Regular measurements of HER-2/neu levels provide a very good indication of the success of chemotherapy and whether a metastasis is receding," he adds. Moreover, after treatment has been completed, the test offers a simple means of monitoring for recurrence of the cancer.

Such benefits are also confirmed by Jean-Pierre Lotz, Chief Medical Oncologist from the Hôpital Tenon at the Université Pierre et Marie Curie in Paris, France. "My first experience with measuring serum HER-2/neu for metastatic breast cancer showed that after patients were given chemotherapy, if the treatment was working, serum levels would rapidly decrease in the first three to four weeks after treatment," he says.

Personalized Diagnostics. The HER-2/neu test also paves the way for the development of increasingly personalized treatment, whereby therapy can be tailored to different types of tumors and diseases in line with an individual patient’s precise needs. The same applies in the case of another biomarker—carbonic anhydrase IX (CA-IX).

CA-IX is an enzyme—a specific type of protein—that is produced in elevated quantities by hypoxic tumors, i.e. those suffering from a poor supply of blood and oxygen. Not only do such tumors generally have a higher resistance to radiation therapy, but they also have been clinically demonstrated to be more aggressive in most cases.

A team headed by Walter Carney at Oncogene Science is currently developing a new in vitro test to detect CA-IX in both tissue and serum. At present, studies are being conducted to verify the procedure.

Meanwhile, at Siemens Healthcare’s Biomarker Research Center in Los Angeles, California, Hartmuth Kolb, PhD, is looking at a related issue. His group is currently developing new positron emission tomography (PET)-based biomarkers that bond to the CA-IX in hypoxic tumors. As a result, the hypoxic tissue shows up as a light-colored area in PET images.

Following detection of CA-IX in a biopsy or serum sample, doctors might then determine the extent of a hypoxic area in the tumor by ordering a PET scan using this new biomarker, or even monitoring the progress of a CA-IX-based treatment.

There are major advantages involved in such an approach. In the future, doctors could use a low-cost lab test from Siemens to examine tissue or serum for CA-IX and thus identify patients with a hypoxic tumor. Such patients would then undergo PET-scanning with the CA-IX-biomarker to determine the location and size of the hypoxic area. They would then be eligible for personalized radiation therapy, or CA-IX-based therapy followed by treatment monitoring based on PET imaging for CA-IX. Since this  combination of procedures is evidence-based, it would ensure that only those patients that genuinely required the procedures would receive them, and that treatment would be optimized for each patient. In that way, Siemens would be making a contribution to raising both the quality and efficiency of patient care.

At present, a number of companies are developing new therapies based on CA-IX markers. However, it will probably be several years before such imaging biomarkers hit the market, since extensive clinical studies must be carried out before approval can be granted.

"It’s true that a single value is not particularly meaningful," says Dr. Robert Krieg, Head of Business Development and Molecular Imaging for Magnetic Resonance Tomography (MR) at Siemens in Erlangen, Germany. "In order to produce genuinely meaningful results, you have to monitor PSA concentrations over time with regular measurements."

Research is therefore focusing on whether special variants of the PSA protein biomarker—the so-called f-PSA, t-PSA, and c-PSA values—might facilitate a more exact diagnosis. Naturally, doctors would like a test with an accuracy of 95 %. But given the diagnostic limitations of PSA tests, doctors almost always resort to biopsies. As a rule, however, this too is a hit-or-miss affair. Although the use of ultrasound to guide the biopsy needle helps, it rarely shows the actual tumor. As a result, doctors often have to take several samples before they can be relatively sure of having hit the suspect area—and even then, in some cases they wind up with false negative results because they’ve missed the tumor  area.

Krieg and his colleagues are therefore working on a new procedure that might be used alongside future f-PSA, t-PSA, and c-PSA tests. The procedure involves a specialized MR imaging technique for examining the prostate gland. A study conducted at the Martha-Maria-Hospital Nuremberg in cooperation with the Waldkrankenhaus Erlangen has already shown that such MR images help doctors target tumors with much greater accuracy when taking a tissue sample.

Known as dynamic contrast-enhanced magnetic resonance imaging or DCE-MRI, (Pictures of the Future, Spring 2007, Research Cooperation Freiburg), this technique is designed to help doctors identify the network of blood vessels that typically form in the tissue around a tumor, thus providing them with much better orientation than a conventional ultrasound image.

But if the f-PSA, t-PSA, and c-PSA tests now under development are successful, they may constitute a powerful-enough decision tool to obviate many biopsies. When, however, their values point to cancer, a urologist will be able to order a DCE-MRI exam to take a closer look. These two techniques would therefore be complementary and altogether important for a precise diagnosis.

Although DCE-MRI is still under development, experience with the HER-2/neu and Troponin-I-Ultra tests demonstrates how lab diagnostics are already helping on a daily basis to identify disease and therefore improve the quality of patient care.
                                                                                                                       Tim Schröder