October 2008
Feature Story

Karen Lusky

Like a kind of internal physiological writing on the wall, Alzheimer’s disease-related markers and signatures seem to appear in the body even before someone develops dementia. If ongoing research studies showing this to be the case continue to pan out, physicians may one day relatively soon have lab tests to help confirm a clinical diagnosis of early AD. They might even be able to identify cognitively normal people at high risk for developing the dementing illness that, as researcher Anne M. Fagan, PhD, of Washington University in St. Louis describes it, “robs you of who you are.”

The availability of such testing would do more than forewarn people that they might need to prepare for long-term care.

Biomarkers represent the “holy grail” of Alzheimer’s disease research, says Brian J. Bacskai, PhD, associate professor of neurology, MassGeneral Institute for Neurodegenerative Diseases, Charlestown, Mass. “If you could get a urine or blood test to give you a yes and no diagnostic answer that was early and sensitive enough, it’d be a whole new world. You could start using clinical trials of drugs to treat people before they developed symptoms.”

The first small steps toward that new world have been taken, though the road isn’t without stumbling blocks. In fact, a biomarker duo for AD—cerebrospinal fluid amyloid beta 1-42 (Aβ42) and taucould be ready for the clinical realm perhaps in one to two years, in the view of John Q. Trojanowski, MD, PhD, professor of pathology and laboratory medicine at the University of Pennsylvania School of Medicine. “We’re getting close,” he says.

The “great promise” of the biomarkers in CSF promoted the Alzheimer’s Neuroimaging Initiative (ADNI), says Dr. Trojanowski, who is also co-director (with Leslie Shaw, PhD) of the ADNI core laboratory housed at the University of Pennsylvania.

ADNI, a five-year natural history, multi-center study of 200 people clinically diagnosed with Alz­heimer’s disease, 200 cognitively normal controls, and 400 participants with mild cognitive impairment, is using a Belgium-based Innogenetics CSF kit for Aβ42 and tau. Every six months, participants in the study receive blood work, neuropsychiatric testing, and brain imaging studies, says Maria Carrillo, PhD, director of medical and scientific relations for the Alzheimer’s Disease Association. CSF testing is done at baseline entry to the study and at the one-year follow­up visit. Final results of the study are expected in 2010, though interim results are coming out all the time, Dr. Carrillo says.

Explaining the theory behind how the CSF biomarker for amyloid and tau works, ADNI core lab co-director Dr. Shaw says that Aβ42, known to be the major component of brain plaques in AD, is one of several peptides derived from the normal breakdown of amyloid precursor protein, or APP. Of all the peptides from APP, he says, Aβ42 is the least soluble, which, in theory, explains why it precipitates out so readily.

The major constituent of tangles in the Alzheimer’s brain is hyperphosphorylated tau. “Tau in its native and normal configuration stabilizes microtubules in neurons,” helping maintain the natural functional structure of nerve cells, Dr. Shaw continues. But that stability disappears when the tau protein is converted to a hyperphosphorylated form, causing the microtubular structure to fall apart. And as the nerve cells degenerate and die, they release their tau protein into the cerebrospinal fluid. The theories for why the hyperphosphorylation of tau occurs in AD are numerous, Dr. Shaw says.

The Innogenetics test used in the ADNI study includes Aβ42, phospho-tau 181 (which means it’s phosphorylated at the 181 site of the tau molecule), and total tau protein, says John Lawson, U.S. market development manager for Innogenetics. To obtain the highest sensitivity and specificity of 85 percent or higher for AD, you need all three biomarkers, Lawson says. For the ADNI study, the company has combined the three tests into one multiparameter assay using the xMAP platform from Luminex Corp.

The Innogenetics test shows good ability to predict who among those with mild cognitive impairment can be expected to develop AD, says Dirk Wout­ers, international mark­eting manager for Innogenetics. But “we see very early signs that the CSF biomarker profile—low amyloid-beta 1-42 together with high phospho-tau and total tau—is there even before people have MCI. The profile doesn’t change over the person’s lifetime once it’s evident.”

Other serial biomarkers in the ADNI trial, says the University of Pennsylvania’s Dr. Shaw, include homocysteine; isoprostanes, which are byproducts of oxidative stress; and ApoE genotyping where the ApoE4 genotype is associated with increased risk of AD, he says. ADNI researchers will also perform serial measurements of Aβ42 in study participants’ blood.

Of course, a clinical blood test looking at amyloid levels would be much easier to do compared with a spinal tap, but research findings are thus far more plentiful and robust for the CSF amyloid and tau biomarker. That could change, however, as evidence continues to mount. In fact, Innogenetics, which has a plasma amyloid beta test in the works, presented clinical data on the diagnostic value of its test at the international Clinical Trials on Alzheimer’s Disease meeting last month in France, says Ray Hayduk, general manager for Innogenetics.

Hayduk also points to research published last month in the Proceedings of the National Academy of Sciences that further supports the hypothesis that using plasma amyloid beta as an AD biomarker has merit. The study (which did not use Innogenetics’ research plasma amyloid test) showed that having an elevated level of Aβ42 in the blood significantly increased the risk of developing AD (Schupf N, et al. PNAS. 2008;105:14052–14057; published ahead of print Sept. 8, 2008 at www.pnas.org/content/105/37/14052.abstract).

Led by Richard Mayeux, MD, MSE, of Columbia University, the study measured plasma amyloid beta peptides 1–42 and 1–40 (Aβ40) in 1,125 elderly people residing in northern Manhattan who didn’t have dementia at the initial measurement. People in the highest quartile of Aβ42 on the initial assessment had a 3.5-fold higher risk of developing AD compared with those in the lowest quartile, says lead author Nicole Schupf, PhD, DrPH, a clinical epidemiologist at Columbia University. But when resear­chers repeated the amyloid measurement, on average, 4.5-years later, those whose plasma Aβ42 had declined since the first measurement were the most likely to have developed AD over the 4.5-year followup period. “We didn’t see any relationship between risk of developing AD and Aβ40,” though both of those amyloid peptides tend to rise with age, Dr. Schupf says. The researchers concluded that the declining levels of plasma Aβ42 could indicate that it’s being sequestered in the brain.

“We know that people in families with autosomal dominant early-onset AD who carry mutations in presenilin1, presenilin2, and amyloid precursor protein genes have elevated levels of Aβ42,” Dr. Schupf says. In addition, the first-degree relatives of patients with late-onset AD with no known mutations also show higher Aβ42 levels, she says.

“We need, however, to understand how plasma amyloid beta levels change over the preclinical period,” at disease onset and as the disease progresses, and that will require more followup, Dr. Schupf says.

PET scans that can detect and measure brain amyloid are also in the picture for potentially helping to diagnose AD, and complementing lab biomarkers for the disease.

Participants in the ADNI study receive serial PET scans using what’s known as the Pittsburgh Compound B, or PiB, which has an affinity for amyloid in the brain. The scans allow researchers to visualize and quantify the amyloid plaques, correlating them to the CSF biomarkers for amyloid and tau and other biomarkers, as well as subjects’ progression from cognitively normal to mild cognitive impairment, and from MCI to AD.

Dr. Fagan at Washington University, an ADNI research site, presented findings from an expanded study at the 2008 International Conference on Alzheimer’s Disease in Chicago looking at participants who had the CSF testing and PET PiB imaging two years apart.

“The data as they stand would suggest the amyloid beta value is telling you the same thing as the PiB,” says Dr. Fagan, who is quick to add a but. The expanded study presented at the international conference included 15 people out of the 132 study subjects who had low a-beta in their CSF but were PiB-negative. “But one thing we don’t know is where to draw the cutoff in [Aβ42] values in the CSF, as the levels vary tremendously between people,” she says.

Yet, there may be a disconnect in some people between the two tests that biological differences could explain, she adds.

Another possibility is that amyloid beta 1-42 drops in the CSF prior to amyloid becoming visible on the PiB scan. Dr. Fagan notes that four people in the lower quadrant of amyloid-beta values with negative PiB scans within two years of lumbar puncture have now had another PiB scan. And one is still PiB-negative, and another is now PiB-positive for cortical amyloid. The other two have suggestive evidence of amyloid in select brain regions based on the researchers’ quantitative measures. “Imagers are still working on where one would draw a quantitative cutoff for defining PiB-positive versus PiB-negative, especially in the smaller, independent brain regions,” she says.

The literature has, however, shown “somewhat of a disconnect” between the amount of brain plaques and tangles and the level of dementia in people, Dr. Fagan says. In fact, the century old chicken and egg debate continues about whether the plaques and tangles are the cause or effect of AD.

“Those who don’t buy into the idea that [amyloid beta] is the main culprit in AD cite the fact that ... there is very little correlation between the amount of plaque and cognitive impairment,” says Dr. Fagan. Instead, “the best correlation between cognitive impairment and pathology is based on how many synapses a person still has [or] synapse density at death.”

Dr. Bacskai at Mass. General Institute for Neurodegenerative Diseases predicts studies underway now and that will begin in the next couple of years will at least answer the question of whet­her the amount of plaque correlates to dementia.

He is in the “amyloid is bad” camp. Research performed by Dr. Bacskai’s lab on mice with senile plaques suggests that the amyloid plaques came first, followed by dysregulation of intracellular calcium levels in neurons and disruption in the neuronal signaling network.

He sees a model for AD where something happens that makes the body produce too much of the amyloid-beta in the brain or reduces clearance of amyloid-beta, creating an accumulation.

One interesting biomarker in early development, in fact, appears to be a defect in monocytes in people with AD that prevents them from clearing amyloid-beta.

“In AD research, there are a lot of ideas about how to skin the cat. We believe that sporadic AD is at least two diseases, one with a relatively understood mechanism—defective innate immune function of amyloid-beta phagocytosis and a second, not yet understood,” says Miodrag Micic, PhD, MTM, vice president of research and development for MP Biomedicals, Santa Ana, Calif., which is developing a monocytebased AD blood test. The work is based on research being done in the lab of Milan Fiala, MD, research professor at the University of California at Los Angeles Orthopaedic Hospital.

Dr. Fiala and his team first developed a test looking at the amyloid-clearing ability of macro­phages (macro­phages differentiate from monocytes when they enter tissues like lung, liver, or brain, and also in a test tube in a special medium after 10-day incubation). The test is labor-intensive, although it has advantages for testing anti-amyloid-beta drugs. To make the diagnostic test practical, they came up with a rapid 24-hour flow cytometry test with fresh blood-derived monocytes that MP Biomedicals has turned into a kit it hopes to get cleared by the FDA for clinical use.

Using the kit, Dr. Micic explains, “you draw a patient’s blood, separate the monocytes, and culture them overnight” with fluorescently labeled amyloid-beta (1–42). “Afterward,” he says, “you wash and stain the macro­phages and do analysis by flow cytometry or under a fluorescent or confocal microscope.”

Viewed under the fluorescent microscope, macrophages from people with clinically diagnosed AD do not transport amyloid-beta into an intracellular location, but instead retain it on the cell surface, Dr. Micic says. But if macro­phages from control subjects are looked at, fluorescently labeled amyloid-beta is transported into intracellular locations such as endosomes and lysosomes.

Using the flow cytometric test, thus far, says UCLA’s Dr. Fiala, 92 percent of people with clinically diagnosed AD are under 400 mean fluorescence intensity units (mean 179) on the monocyte test. “If we use a control group of cognitively high-achieving professors of similar age, then the results are all above 500 with a mean of 1,449 mean fluorescence units,” he says. But about 40 to 50 percent of a control group that is of similar age as the AD group show abnormal results. “To us, those findings are consistent with what’s known to be the percentage of people who by age 85 in the general population will have AD.”

Preliminary study data suggest that the flow cytometric test is abnormal in 60 percent of patients with mild cognitive impairment. “The hope is that prospective studies will determine whether the test will predict the risk of progression from MCI to AD,” Dr. Fiala says.

He will even go so far as to say that the monocyte test might identify people early in life who have the immune defect, since “we have found that in several families, unaffected blood relatives have a similar defect as the index case.” To put this in perspective, a previous study found that nuns who wrote autobiographical essays at age 22, and whose brains were tested and shown to have AD when they died, had indications of cognitive abnormalities in their writing at age 22. “It’s possible that cognitive and immune defects start very early in life,” he says.

Why, in some people, might monocytes not be able to clear amyloid? “That’s the big question and one to which we don’t yet have an answer,” Dr. Fiala says. But “we know their function is impaired at the transcription level with respect to the genes important in immune function.”

And when viewed under the microscope, the macrophages look “tired,” and smaller than normal, lacking the surface structures necessary for phagocytosis, Dr. Fiala says. “We think the reason is complex” and could have a lot to do with aging, genetics, dietary factors, viruses, and toxins—“and who knows what.”

Dr. Fiala and his team are doing studies to identify the factors. He notes, however, that some researchers think the monocyte impairment could be related to deficits of hematopoetic factors, which could be helped by existing hematopoietic treatments that boost bone marrow production.

“But we think some specific factors are responsible for AD because not everyone gets it, even in very old age. We have tested one of the oldest people in California a 109 year old person who still played bridge and she was okay as far as her immune system’s ability to clear amyloid.”

Research testing a potential AD test composed of 18 plasma communication or signaling factors may support some of what Dr. Fiala is finding, says Tony Wyss-Coray, PhD, associate professor of neurology at Stanford, one of the researchers developing the proteomic panel and cofounder of Satoris Inc., Redwood City, Calif., which is commercializing the test.

The communication factors involve immune responses, in addition to hematopoiesis, apoptosis, and neuronal support, according to the study involving 259 subjects, published last year in Nature Medicine (Ray S, et al. Nat Med. 2007;13 [ 11]: 1359–13 62; www.ncbi.nlm.nih.gov/pubmed/17934472).

“Dr. Fiala observes that the macrophage type cells are not working as well in people with AD as in controls,” says Dr. Wyss-Coray. And in the research published in Nature Medicine, Dr. Wyss-Coray and his colleagues found a reduction in several communication factors involved in generating new macro­phages or attracting macro­phages, allowing them to enter tissues, for example.

“So maybe there are not enough of these factors being produced and that’s why macrophages are not effective in older individuals,” he says. As for why the communication factors are reduced, “at this point, we really don’t know.”

But “what we have found could be related to inflammation,” he adds, noting that the first part of the inflammatory response is obviously helpful in that it attempts to remove a pathogen or injury, for example. But if the inflammatory response continues, it can lead to tissue damage. “Maybe that’s what is happening in AD. The same cells initially involved in removing amyloid and trying to maintain the brain don’t work as well in some individuals, and clearly they don’t with aging. Perhaps they get overloaded and start to produce toxic proteins or it could be specific factors such as cytokines. Tumor necrosis factor can be protective but also toxic under other circumstances. Like everything in biology or life, it depends on the dose.”

How well did the proteomic panel identify people with clinically diagnosed AD? Using a statistical algorithm to develop breakpoints predicting the likelihood that a person is positive or negative, about 90 percent were positive on the test in the people with AD, says Dr. Wyss-Coray. “About 10 percent were positive in the control group,” he adds.

“Perhaps the people in the control group did have MCI and we didn’t know it, or perhaps the people who tested negative in the AD group had some other type of dementia. We don’t know that.”

Satoris is planning to follow people over time or even confirm after death that they did or did not have AD, Dr. Wyss-Coray says.

Cristopher McReynolds, CEO of Satoris, says that in one part of the study published in Nature Medicine, researchers used the test on people with mild cognitive impairment and “with a high level of accuracy [91 percent, according to the study report], were able to detect the early stages of the AD process, anywhere from two to six years before a clinician could detect it.”

Power3 Medical Products Inc., The Woodlands, Tex., is also in the early stages of developing a proteomic test, NuroPro, that monitors the concentration of a series of protein biomarkers in the blood serum to differentiate among various neurodegenerative diseases that cause dementia.

“We have discovered that a group of proteinsa total of 59 for Alzheimer’s, Parkinson’s, and Lou Gehrig’s [ALS] diseases—deviate in their concentrations from normal ranges in statistically significant, specific ways,” says Ira L. Goldknopf, PhD, director of proteomics for Power3 Medical Products.

Dr. Goldknopf declined to name the proteins because the company is in the process of filing patent applications. He does say the biomarkers reflect various aspects of the neurodegenerative process, “including inflammation, differences in the ability of the cells to protect themselves from outside destructive forces, as well as indications of destructive internal events in the neuronal cells of the patients.”

Each disease has its own protein biomarker profile that has things in common with, and that differ from, the other diseases, he says.

“If a person has a mild cognitive impairment, this test might say it doesn’t look like AD but there is some indication that it’s Lewy body or another type of dementia, or it might indicate an early form of AD,” Dr. Goldknopf says.

“We have indications that some of our biomarkers display differential expressions that correlate with disease severity, and that early AD appears somewhat different from later stages. Further testing should flesh this out.”

Sometimes the testing shows a person has no indications of any of the dementias, meaning that one should look for other causes of the person’s cognitive problems, says Dr. Goldknopf. “There are all kinds of reasons for mildly impaired cognition. The question is, are we seeing neurodegeneration? That’s what’s really key. The test gives objective, quantifiable answers, and it has probabilities built in, too, that someone has a diagnosis.”

Dr. Goldknopf says the test will have sensitivity and specificity in the 90 percent range, though it’s too early to be precise. “It’s still a work in progress, but in our experience, when you are on the right track, the test gets better with time.”

Washington University’s Dr. Fagan believes that biomarkers in development reflect different aspects of AD. She predicts we will eventually end up with a panel of biomarkers that provide an idea of where a person is in the disease process.

Biomarkers are helpful in providing a more definitive answer about whether a person has AD or appears to be on track for developing it, but the ultimate goal, of course, is to couple biomarkers with disease-modifying treatments that can delay or prevent onset.

Dr. Fiala at UCLA, which is working on the monocyte-based test, says his lab has “one ace up its sleeve” in the treatment arena: Indian curry, which the lab is now testing in vitro. “The research shows that patients who have a problem clearing amyloid are helped in test tubes by adding curcumin,” an active ingredient in curry. “But this is ... in the test tube. We hope our test will respond in vivo to curcumin.”

Biomarkers that can identify people earlier in the AD process may help give anti-amyloid drugs, which thus far haven’t shown much success, a better shot at working. Todd E. Golde, MD, PhD, a professor and chair of the Department of Neuroscience at Mayo Clinic in Jacksonville, Fla., compares treating people with full-blown AD with anti-amyloid drugs to giving a statin to someone with atherosclerotic disease who has had four myocardial infarctions and is in heart failure. “It’s not likely to work very well at that point but it might work well in prevention.”

Drug-maker Eli Lilly and Company is using CSF and plasma amyloid biomarkers in clinical trials testing two anti-amyloid products: a gamma-secretase inhibitor that inhibits amyloid-beta production, which is in phase three clinical trials, and a monoclonal antibody targeting amyloid-beta, which has completed phase two trials.

“In phase two studies,” says neurologist Eric Siem­ers, MD, medical director for the AD research team at Lilly, “biomarker test­ing showed that giving the highest dose used in phase three trials of the gamma-secretase inhibitor ... lowered the amount of amyloid-beta in the plasma by over 70 percent.”

In phase two studies, the monoclonal antibody against amyloidbeta resulted in increased amounts of amyloid-beta peptides bound (as opposed to unbound) to the antibody in plasma and spinal fluid. As expected, unbound amyloid-beta 1–40 in the spinal fluid decreased. But unbound amyloid-beta 1–42 (the form found in amyloid plaques) increased in spinal fluid. “Our interpretation of that is that the plaques are dissolving some, leading to a higher level of unbound amyloid-beta 1–42 in the CSF,” Dr. Siemers says.

“In the future,” says Cameron Durrant, MD, MBA, on the board of directors for emerging biopharmaceutical company Anavex Life Sciences Corp., Athens, Greece, “there could be drugs for AD that have different mechanisms but produce the same outcome and prevent progression in people who have the condition or prevent people from developing it at all.” Anavex is in the preclinical stage of developing a drug that interferes upstream with the reactive oxygen species that can cause oxidative stress.

Finding magic bullets for AD pathology, however, might not help a person who has AD and other causes of dementia at the same time. For example, Mayo’s Dr. Golde notes that although vascular dementia is considered to be a separate disease process, some dementias overlap.

He points to a presentation at the international Alzheimer’s conference in July by neuropathologist Dennis Dickson, MD, professor of neuroscience, Mayo Clinic Jacksonville, that addressed the mixed brain pathology seen at autopsy in some people with dementia. For example, Dr. Golde says, TDP-43, a genetically validated cause of ALS, is “the new kid on the block.” And TDP pathology is also seen in 20 percent of AD cases, so it’s evident that subtypes of AD at the level of pathology may exist, he adds.

Dr. Dickson has a few caveats to throw in the debate about finding specific AD biomarkers and disease-modifying treatments.

“Many people with a clinical diagnosis of AD actually have more than one pathology seen at autopsy, most often cerebrovascular disease or Lewy body pathology, but there are others,” says Dr. Dickson, professor of laboratory medicine and pathology at Mayo. This is even more true of people the older they get.

“So in terms of biomarkers or even treatment, you may get a mixed response in those individuals,” he cautions. Dr. Dickson also says it remains to be seen if cognitively normal people with amyloid in their brains seen on imaging will all convert to AD. “It may just be a feature of one type of brain aging.”

George Perry, PhD, editor-in-chief of the Journal of Alzheimer’s Disease (www.j-alz.com) and dean of the College of Sciences at the University of Texas at San Antonio, says more work needs to be done to develop biomarkers with greater than 90 percent specificity for AD, to compete effectively with current clinical tests. “To come up with better biomarkers will require a new understanding of the aging process and how neurons adapt during aging,” he says.

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