Posts Tagged ‘mass spectroscopy’

Clinical Laboratory Challenges

Larry H. Bernstein, MD, FCAP, Curator




The Lab and CJD: Safe Handling of Infectious Prion Proteins

Body fluids from individuals with possible Creutzfeldt-Jakob disease (CJD) present distinctive safety challenges for clinical laboratories. Sporadic, iatrogenic, and familial CJD (known collectively as classic CJD), along with variant CJD, kuru, Gerstmann-Sträussler-Scheinker, and fatal familial insomnia, are prion diseases, also known as transmissible spongiform encephalopathies. Prion diseases affect the central nervous system, and from the onset of symptoms follow a typically rapid progressive neurological decline. While prion diseases are rare, it is not uncommon for the most prevalent form—sporadic CJD—to be included in the differential diagnosis of individuals presenting with rapid cognitive decline. Thus, laboratories may deal with a significant number of possible CJD cases, and should have protocols in place to process specimens, even if a confirmatory diagnosis of CJD is made in only a fraction of these cases.

The Lab’s Role in Diagnosis

Laboratory protocols for handling specimens from individuals with possible, probable, and definitive cases of CJD are important to ensure timely and appropriate patient management. When the differential includes CJD, an attempt should be made to rule-in or out other causes of rapid neurological decline. Laboratories should be prepared to process blood and cerebrospinal fluid (CSF) specimens in such cases for routine analyses.

Definitive diagnosis requires identification of prion aggregates in brain tissue, which can be achieved by immunohistochemistry, a Western blot for proteinase K-resistant prions, and/or by the presence of prion fibrils. Thus, confirmatory diagnosis is typically achieved at autopsy. A probable diagnosis of CJD is supported by elevated concentration of 14-3-3 protein in CSF (a non-specific marker of neurodegeneration), EEG, and MRI findings. Thus, the laboratory may be required to process and send CSF samples to a prion surveillance center for 14-3-3 testing, as well as blood samples for sequencing of the PRNP gene (in inherited cases).

Processing Biofluids

Laboratories should follow standard protective measures when working with biofluids potentially containing abnormally folded prions, such as donning standard personal protective equipment (PPE); avoiding or minimizing the use of sharps; using single-use disposable items; and processing specimens to minimize formation of aerosols and droplets. An additional safety consideration is the use of single-use disposal PPE; otherwise, re-usable items must be either cleaned using prion-specific decontamination methods, or destroyed.

Blood. In experimental models, infectivity has been detected in the blood; however, there have been no cases of secondary transmission of classical CJD via blood product transfusions in humans. As such, blood has been classified, on epidemiological evidence by the World Health Organization (WHO), as containing “no detectible infectivity,” which means it can be processed by routine methods. Similarly, except for CSF, all other body fluids contain no infectivity and can be processed following standard procedures.

In contrast to classic CJD, there have been four cases of suspected secondary transmission of variant CJD via transfused blood products in the United Kingdom. Variant CJD, the prion disease associated with mad cow disease, is unique in its distribution of prion aggregates outside of the central nervous system, including the lymph nodes, spleen, and tonsils. For regions where variant CJD is a concern, laboratories should consult their regulatory agencies for further guidance.

CSF. Relative to highly infectious tissues of the brain, spinal cord, and eye, infectivity has been identified less often in CSF and is considered to have “low infectivity,” along with kidney, liver, and lung tissue. Since CSF can contain infectious material, WHO has recommended that analyses not be performed on automated equipment due to challenges associated with decontamination. Laboratories should perform a risk assessment of their CSF processes, and, if deemed necessary, consider using manual methods as an alternative to automated systems.


The infectious agent in prion disease is unlike any other infectious pathogen encountered in the laboratory; it is formed of misfolded and aggregated prion proteins. This aggregated proteinacious material forms the infectious unit, which is incredibly resilient to degradation. Moreover, in vitro studies have demonstrated that disrupting large aggregates into smaller aggregates increases cytotoxicity. Thus, if the aim is to abolish infectivity, all aggregates must be destroyed. Disinfectant procedures used for viral, bacterial, and fungal pathogens such as alcohol, boiling, formalin, dry heat (<300°C), autoclaving at 121°C for 15 minutes, and ionizing, ultraviolet, or microwave radiation, are either ineffective or variably effective against aggregated prions.

The only means to ensure no risk of residual infectious prions is to use disposable materials. This is not always practical, as, for instance, a biosafety cabinet cannot be discarded if there is a CSF spill in the hood. Fortunately, there are several protocols considered sufficient for decontamination. For surfaces and heat-sensitive instruments, such as a biosafety cabinet, WHO recommends flooding the surface with 2N NaOH or undiluted NaClO, letting stand for 1 hour, mopping up, and rinsing with water. If the surface cannot tolerate NaOH or NaClO, thorough cleaning will remove most infectivity by dilution. Laboratories may derive some additional benefit by using one of the partially effective methods discussed previously. Non-disposable heat-resistant items preferably should be immersed in 1N NaOH, heated in a gravity displacement autoclave at 121°C for 30 min, cleaned and rinsed in water, then sterilized by routine methods. WHO has outlined several alternate decontamination methods. Using disposable cover sheets is one simple solution to avoid contaminating work surfaces and associated lengthy decontamination procedures.

With standard PPE—augmented by a few additional safety measures and prion-specific decontamination procedures—laboratories can safely manage biofluid testing in cases of prion disease.


The Microscopic World Inside Us  

Emerging Research Points to Microbiome’s Role in Health and Disease

Thousands of species of microbes—bacteria, viruses, fungi, and protozoa—inhabit every internal and external surface of the human body. Collectively, these microbes, known as the microbiome, outnumber the body’s human cells by about 10 to 1 and include more than 1,000 species of microorganisms and several million genes residing in the skin, respiratory system, urogenital, and gastrointestinal tracts. The microbiome’s complicated relationship with its human host is increasingly considered so crucial to health that researchers sometimes call it “the forgotten organ.”

Disturbances to the microbiome can arise from nutritional deficiencies, antibiotic use, and antiseptic modern life. Imbalances in the microbiome’s diverse microbial communities, which interact constantly with cells in the human body, may contribute to chronic health conditions, including diabetes, asthma and allergies, obesity and the metabolic syndrome, digestive disorders including irritable bowel syndrome (IBS), and autoimmune disorders like multiple sclerosis and rheumatoid arthritis, research shows.

While study of the microbiome is a growing research enterprise that has attracted enthusiastic media attention and venture capital, its findings are largely preliminary. But some laboratorians are already developing a greater appreciation for the microbiome’s contributions to human biochemistry and are considering a future in which they expect to measure changes in the microbiome to monitor disease and inform clinical practice.

Pivot Toward the Microbiome

Following the National Institutes of Health (NIH) Human Genome Project, many scientists noted the considerable genetic signal from microbes in the body and the existence of technology to analyze these microorganisms. That realization led NIH to establish the Human Microbiome Project in 2007, said Lita Proctor, PhD, its program director. In the project’s first phase, researchers studied healthy adults to produce a reference set of microbiomes and a resource of metagenomic sequences of bacteria in the airways, skin, oral cavities, and the gastrointestinal and vaginal tracts, plus a catalog of microbial genome sequences of reference strains. Researchers also evaluated specific diseases associated with disturbances in the microbiome, including gastrointestinal diseases such as Crohn’s disease, ulcerative colitis, IBS, and obesity, as well as urogenital conditions, those that involve the reproductive system, and skin diseases like eczema, psoriasis, and acne.

Phase 1 studies determined the composition of many parts of the microbiome, but did not define how that composition affects health or specific disease. The project’s second phase aims to “answer the question of what microbes actually do,” explained Proctor. Researchers are now examining properties of the microbiome including gene expression, protein, and human and microbial metabolite profiles in studies of pregnant women at risk for preterm birth, the gut hormones of patients at risk for IBS, and nasal microbiomes of patients at risk for type 2 diabetes.

Promising Lines of Research

Cystic fibrosis and microbiology investigator Michael Surette, PhD, sees promising microbiome research not just in terms of evidence of its effects on specific diseases, but also in what drives changes in the microbiome. Surette is Canada research chair in interdisciplinary microbiome research in the Farncombe Family Digestive Health Research Institute at McMaster University
in Hamilton, Ontario.

One type of study on factors driving microbiome change examines how alterations in composition and imbalances in individual patients relate to improving or worsening disease. “IBS, cystic fibrosis, and chronic obstructive pulmonary disease all have periods of instability or exacerbation,” he noted. Surette hopes that one day, tests will provide clinicians the ability to monitor changes in microbial composition over time and even predict when a patient’s condition is about to deteriorate. Monitoring perturbations to the gut microbiome might also help minimize collateral damage to the microbiome during aggressive antibiotic therapy for hospitalized patients, he added.

Monitoring changes to the microbiome also might be helpful for “culture negative” patients, who now may receive multiple, unsuccessful courses of different antibiotics that drive antibiotic resistance. Frustration with standard clinical biology diagnosis of lung infections in cystic fibrosis patients first sparked Surette’s investigations into the microbiome. He hopes that future tests involving the microbiome might also help asthma patients with neutrophilia, community-acquired pneumonia patients who harbor complex microbial lung communities lacking obvious pathogens, and hospitalized patients with pneumonia or sepsis. He envisions microbiome testing that would look for short-term changes indicating whether or not a drug is effective.

Companion Diagnostics

Daniel Peterson, MD, PhD, an assistant professor of pathology at Johns Hopkins University School of Medicine in Baltimore, believes the future of clinical testing involving the microbiome lies in companion diagnostics for novel treatments, and points to companies that are already developing and marketing tests that will require such assays.

Examples of microbiome-focused enterprises abound, including Genetic Analysis, based in Oslo, Norway, with its high-throughput test that uses 54 probes targeted to specific bacteria to measure intestinal gut flora imbalances in inflammatory bowel disease and irritable bowel syndrome patients. Paris, France-based Enterome is developing both novel drugs and companion diagnostics for microbiome-related diseases such as IBS and some metabolic diseases. Second Genome, based in South San Francisco, has developed an experimental drug, SGM-1019, that the company says blocks damaging activity of the microbiome in the intestine. Cambridge, Massachusetts-based Seres Therapeutics has received Food and Drug Administration orphan drug designation for SER-109, an oral therapeutic intended to correct microbial imbalances to prevent recurrent Clostridium difficile infection in adults.

One promising clinical use of the microbiome is fecal transplantation, which both prospective and retrospective studies have shown to be effective in patients with C. difficile infections who do not respond to front-line therapies, said James Versalovic, MD, PhD, director of Texas Children’s Hospital Microbiome Center and professor of pathology at Baylor College of Medicine in Houston. “Fecal transplants and other microbiome replacement strategies can radically change the composition of the microbiome in hours to days,” he explained.

But NIH’s Proctor discourages too much enthusiasm about fecal transplant. “Natural products like stool can have [side] effects,” she pointed out. “The [microbiome research] field needs to mature and we need to verify outcomes before anything becomes routine.”

Hurdles for Lab Testing

While he is hopeful that labs someday will use the microbiome to produce clinically useful information, Surette pointed to several problems that must be solved beforehand. First, molecular methods commonly used right now should be more quantitative and accurate. Additionally, research on the microbiome encompasses a wide variety of protocols, some of which are better at extracting particular types of bacteria and therefore can give biased views of communities living in the body. Also, tests may need to distinguish between dead and live microbes. Another hurdle is that labs using varied bioinfomatic methods may produce different results from the same sample, a problem that Surette sees as ripe for a solution from clinical laboratorians, who have expertise in standardizing robust protocols and in automating tests.

One way laboratorians can prepare for future, routine microbiome testing is to expand their notion of clinical chemistry to include both microbial and human biochemistry. “The line between microbiome science and clinical science is blurring,” said Versalovic. “When developing future assays to detect biochemical changes in disease states, we must consider the contributions of microbial metabolites and proteins and how to tailor tests to detect them.” In the future, clinical labs may test for uniquely microbial metabolites in various disease states, he predicted.


Automated Review of Mass Spectrometry Results  

Can We Achieve Autoverification?

Author: Katherine Alexander and Andrea R. Terrell, PhD  // Date: NOV.1.2015  // Source:Clinical Laboratory News



Paralleling the upswing in prescription drug misuse, clinical laboratories are receiving more requests for mass spectrometry (MS) testing as physicians rely on its specificity to monitor patient compliance with prescription regimens. However, as volume has increased, reimbursement has declined, forcing toxicology laboratories both to increase capacity and lower their operational costs—without sacrificing quality or turnaround time. Now, new solutions are available enabling laboratories to bring automation to MS testing and helping them with the growing demand for toxicology and other testing.

What is the typical MS workflow?

A typical workflow includes a long list of manual steps. By the time a sample is loaded onto the mass spectrometer, it has been collected, logged into the lab information management system (LIMS), and prepared for analysis using a variety of wet chemistry techniques.

Most commercial clinical laboratories receive enough samples for MS analysis to batch analyze those samples. A batch consists of a calibrator(s), quality control (QC) samples, and patient/donor samples. Historically, the method would be selected (i.e. “analysis of opiates”), sample identification information would be entered manually into the MS software, and the instrument would begin analyzing each sample. Upon successful completion of the batch, the MS operator would view all of the analytical data, ensure the QC results were acceptable, and review each patient/donor specimen, looking at characteristics such as peak shape, ion ratios, retention time, and calculated concentration.

The operator would then post acceptable results into the LIMS manually or through an interface, and unacceptable results would be rescheduled or dealt with according to lab-specific protocols. In our laboratory we perform a final certification step for quality assurance by reviewing all information about the batch again, prior to releasing results for final reporting through the LIMS.

What problems are associated with this workflow?

The workflow described above results in too many highly trained chemists performing manual data entry and reviewing perfectly acceptable analytical results. Lab managers would prefer that MS operators and certifying scientists focus on troubleshooting problem samples rather than reviewing mounds of good data. Not only is the current process inefficient, it is mundane work prone to user errors. This risks fatigue, disengagement, and complacency by our highly skilled scientists.

Importantly, manual processes also take time. In most clinical lab environments, turnaround time is critical for patient care and industry competitiveness. Lab directors and managers are looking for solutions to automate mundane, error-prone tasks to save time and costs, reduce staff burnout, and maintain high levels of quality.

How can software automate data transfer from MS systems to LIMS?

Automation is not a new concept in the clinical lab. Labs have automated processes in shipping and receiving, sample preparation, liquid handling, and data delivery to the end user. As more labs implement MS, companies have begun to develop special software to automate data analysis and review workflows.

In July 2011, AIT Labs incorporated ASCENT into our workflow, eliminating the initial manual peak review step. ASCENT is an algorithm-based peak picking and data review system designed specifically for chromatographic data. The software employs robust statistical and modeling approaches to the raw instrument data to present the true signal, which often can be obscured by noise or matrix components.

The system also uses an exponentially modified Gaussian (EMG) equation to apply a best-fit model to integrated peaks through what is often a noisy signal. In our experience, applying the EMG results in cleaner data from what might appear to be poor chromatography ultimately allows us to reduce the number of samples we might otherwise rerun.

How do you validate the quality of results?

We’ve developed a robust validation protocol to ensure that results are, at minimum, equivalent to results from our manual review. We begin by building the assay in ASCENT, entering assay-specific information from our internal standard operating procedure (SOP). Once the assay is configured, validation proceeds with parallel batch processing to compare results between software-reviewed data and staff-reviewed data. For new implementations we run eight to nine batches of 30–40 samples each; when we are modifying or upgrading an existing implementation we run a smaller number of batches. The parallel batches should contain multiple positive and negative results for all analytes in the method, preferably spanning the analytical measurement range of the assay.

The next step is to compare the results and calculate the percent difference between the data review methods. We require that two-thirds of the automated results fall within 20% of the manually reviewed result. In addition to validating patient sample correlation, we also test numerous quality assurance rules that should initiate a flag for further review.

What are the biggest challenges during implementation and continual improvement initiatives?

On the technological side, our largest hurdle was loading the sequence files into ASCENT. We had created an in-house mechanism for our chemists to upload the 96-well plate map for their batch into the MS software. We had some difficulty transferring this information to ASCENT, but once we resolved this issue, the technical workflow proceeded fairly smoothly.

The greater challenge was changing our employees’ mindset from one of fear that automation would displace them, to a realization that learning this new technology would actually make them more valuable. Automating a non-mechanical process can be a difficult concept for hands-on scientists, so managers must be patient and help their employees understand that this kind of technology leverages the best attributes of software and people to create a powerful partnership.

We recommend that labs considering automated data analysis engage staff in the validation and implementation to spread the workload and the knowledge. As is true with most technology, it is best not to rely on just one or two super users. We also found it critical to add supervisor level controls on data file manipulation, such as removing a sample that wasn’t run from the sequence table. This can prevent inadvertent deletion of a file, requiring reinjection of the entire batch!


Understanding Fibroblast Growth Factor 23

Author: Damien Gruson, PhD  // Date: OCT.1.2015  // Source: Clinical Laboratory News


What is the relationship of FGF-23 to heart failure?

A Heart failure (HF) is an increasingly common syndrome associated with high morbidity, elevated hospital readmission rates, and high mortality. Improving diagnosis, prognosis, and treatment of HF requires a better understanding of its different sub-phenotypes. As researchers gained a comprehensive understanding of neurohormonal activation—one of the hallmarks of HF—they discovered several biomarkers, including natriuretic peptides, which now are playing an important role in sub-phenotyping HF and in driving more personalized management of this chronic condition.

Like the natriuretic peptides, fibroblast growth factor 23 (FGF-23) could become important in risk-stratifying and managing HF patients. Produced by osteocytes, FGF-23 is a key regulator of phosphorus homeostasis. It binds to renal and parathyroid FGF-Klotho receptor heterodimers, resulting in phosphate excretion, decreased 1-α-hydroxylation of 25-hydroxyvitamin D, and decreased parathyroid hormone (PTH) secretion. The relationship to PTH is important because impaired homeostasis of cations and decreased glomerular filtration rate might contribute to the rise of FGF-23. The amino-terminal portion of FGF-23 (amino acids 1-24) serves as a signal peptide allowing secretion into the blood, and the carboxyl-terminal portion (aa 180-251) participates in its biological action.

How might FGF-23 improve HF risk assessment?

Studies have shown that FGF-23 is related to the risk of cardiovascular diseases and mortality. It was first demonstrated that FGF-23 levels were independently associated with left ventricular mass index and hypertrophy as well as mortality in patients with chronic kidney disease (CKD). FGF-23 also has been associated with left ventricular dysfunction and atrial fibrillation in coronary artery disease subjects, even in the absence of impaired renal function.

FGF-23 and FGF receptors are both expressed in the myocardium. It is possible that FGF-23 has direct effects on the heart and participates in the physiopathology of cardiovascular diseases and HF. Experiments have shown that for in vitro cultured rat cardiomyocytes, FGF-23 stimulates pathological hypertrophy by activating the calcineurin-NFAT pathway—and in wild-type mice—the intra-myocardial or intravenous injection of FGF-23 resulted in left ventricular hypertrophy. As such, FGF-23 appears to be a potential stimulus of myocardial hypertrophy, and increased levels may contribute to the worsening of heart failure and long-term cardiovascular death.

Researchers have documented that HF patients have elevated FGF-23 circulating levels. They have also found a significant correlation between plasma levels of FGF-23 and B-type natriuretic peptide, a biomarker related to ventricular stretch and cardiac hypertrophy, in patients with left ventricular hypertrophy. As such, measuring FGF-23 levels might be a useful tool to predict long-term adverse cardiovascular events in HF patients.

Interestingly, researchers have documented a significant relationship between FGF-23 and PTH in both CKD and HF patients. As PTH stimulates FGF-23 expression, it could be that in HF patients, increased PTH levels increase the bone expression of FGF-23, which enhances its effects on the heart.


The Past, Present, and Future of Western Blotting in the Clinical Laboratory

Author: Curtis Balmer, PhD  // Date: OCT.1.2015  // Source: Clinical Laboratory News


Much of the discussion about Western blotting centers around its performance as a biological research tool. This isn’t surprising. Since its introduction in the late 1970s, the Western blot has been adopted by biology labs of virtually every stripe, and become one of the most widely used techniques in the research armamentarium. However, Western blotting has also been employed in clinical laboratories to aid in the diagnosis of various diseases and disorders—an equally important and valuable application. Yet there has been relatively little discussion of its use in this context, or of how advances in Western blotting might affect its future clinical use.

Highlighting the clinical value of Western blotting, Stanley Naides, MD, medical director of Immunology at Quest Diagnostics observed that, “Western blotting has been a very powerful tool in the laboratory and for clinical diagnosis. It’s one of many various methods that the laboratorian brings to aid the clinician in the diagnosis of disease, and the selection and monitoring of therapy.” Indeed, Western blotting has been used at one time or the other to aid in the diagnosis of infectious diseases including hepatitis C (HCV), HIV, Lyme disease, and syphilis, as well as autoimmune disorders such as paraneoplastic disease and myositis conditions.

However, Naides was quick to point out that the choice of assays to use clinically is based on their demonstrated sensitivity and performance, and that the search for something better is never-ending. “We’re constantly looking for methods that improve detection of our target [protein],” Naides said. “There have been a number of instances where we’ve moved away from Western blotting because another method proves to be more sensitive.” But this search can also lead back to Western blotting. “We’ve gone away from other methods because there’s been a Western blot that’s been developed that’s more sensitive and specific. There’s that constant movement between methods as new tests are developed.”

In recent years, this quest has been leading clinical laboratories away from Western blotting toward more sensitive and specific diagnostic assays, at least for some diseases. Using confirmatory diagnosis of HCV infection as an example, Sai Patibandla, PhD, director of the immunoassay group at Siemens Healthcare Diagnostics, explained that movement away from Western blotting for confirmatory diagnosis of HCV infection began with a technical modification called Recombinant Immunoblotting Assay (RIBA). RIBA streamlines the conventional Western blot protocol by spotting recombinant antigen onto strips which are used to screen patient samples for antibodies against HCV. This approach eliminates the need to separate proteins and transfer them onto a membrane.

The RIBA HCV assay was initially manufactured by Chiron Corporation (acquired by Novartics Vaccines and Diagnostics in 2006). It received Food and Drug Administration (FDA) approval in 1999, and was marketed as Chiron RIBA HCV 3.0 Strip Immunoblot Assay. Patibandla explained that, at the time, the Chiron assay “…was the only FDA-approved confirmatory testing for HCV.” In 2013 the assay was discontinued and withdrawn from the market due to reports that it was producing false-positive results.

Since then, clinical laboratories have continued to move away from Western blot-based assays for confirmation of HCV in favor of the more sensitive technique of nucleic acid testing (NAT). “The migration is toward NAT for confirmation of HCV [diagnosis]. We don’t use immunoblots anymore. We don’t even have a blot now to confirm HCV,” Patibandla said.

Confirming HIV infection has followed a similar path. Indeed, in 2014 the Centers for Disease Control and Prevention issued updated recommendations for HIV testing that, in part, replaced Western blotting with NAT. This change was in response to the recognition that the HIV-1 Western blot assay was producing false-negative or indeterminable results early in the course of HIV infection.

At this juncture it is difficult to predict if this trend away from Western blotting in clinical laboratories will continue. One thing that is certain, however, is that clinicians and laboratorians are infinitely pragmatic, and will eagerly replace current techniques with ones shown to be more sensitive, specific, and effective. This raises the question of whether any of the many efforts currently underway to improve Western blotting will produce an assay that exceeds the sensitivity of currently employed techniques such as NAT.

Some of the most exciting and groundbreaking work in this area is being done by Amy Herr, PhD, a professor of bioengineering at University of California, Berkeley. Herr’s group has taken on some of the most challenging limitations of Western blotting, and is developing techniques that could revolutionize the assay. For example, the Western blot is semi-quantitative at best. This weakness dramatically limits the types of answers it can provide about changes in protein concentrations under various conditions.

To make Western blotting more quantitative, Herr’s group is, among other things, identifying losses of protein sample mass during the assay protocol. About this, Herr explains that the conventional Western blot is an “open system” that involves lots of handling of assay materials, buffers, and reagents that makes it difficult to account for protein losses. Or, as Kevin Lowitz, a senior product manager at Thermo Fisher Scientific, described it, “Western blot is a [simple] technique, but a really laborious one, and there are just so many steps and so many opportunities to mess it up.”

Herr’s approach is to reduce the open aspects of Western blot. “We’ve been developing these more closed systems that allow us at each stage of the assay to account for [protein mass] losses. We can’t do this exactly for every target of interest, but it gives us a really good handle [on protein mass losses],” she said. One of the major mechanisms Herr’s lab is using to accomplish this is to secure proteins to the blot matrix with covalent bonding rather than with the much weaker hydrophobic interactions that typically keep the proteins in place on the membrane.

Herr’s group also has been developing microfluidic platforms that allow Western blotting to be done on single cells, “In our system we’re doing thousands of independent Westerns on single cells in four hours. And, hopefully, we’ll cut that down to one hour over the next couple years.”

Other exciting modifications that stand to dramatically increase the sensitivity, quantitation, and through-put of Western blotting also are being developed and explored. For example, the use of capillary electrophoresis—in which proteins are conveyed through a small electrolyte-filled tube and separated according to size and charge before being dropped onto a blotting membrane—dramatically reduces the amount of protein required for Western blot analysis, and thereby allows Westerns to be run on proteins from rare cells or for which quantities of sample are extremely limited.

Jillian Silva, PhD, an associate specialist at the University of California, San Francisco Helen Diller Family Comprehensive Cancer Center, explained that advances in detection are also extending the capabilities of Western blotting. “With the advent of fluorescence detection we have a way to quantitate Westerns, and it is now more quantitative than it’s ever been,” said Silva.

Whether or not these advances produce an assay that is adopted by clinical laboratories remains to be seen. The emphasis on Western blotting as a research rather than a clinical tool may bias advances in favor of the needs and priorities of researchers rather than clinicians, and as Patibandla pointed out, “In the research world Western blotting has a certain purpose. [Researchers] are always coming up with new things, and are trying to nail down new proteins, so you cannot take Western blotting away.” In contrast, she suggested that for now, clinical uses of Western blotting remain “limited.”


Adapting Next Generation Technologies to Clinical Molecular Oncology Service

Author: Ronald Carter, PhD, DVM  // Date: OCT.1.2015  // Source: Clinical Laboratory News


Next generation technologies (NGT) deliver huge improvements in cost efficiency, accuracy, robustness, and in the amount of information they provide. Microarrays, high-throughput sequencing platforms, digital droplet PCR, and other technologies all offer unique combinations of desirable performance.

As stronger evidence of genetic testing’s clinical utility influences patterns of patient care, demand for NGT testing is increasing. This presents several challenges to clinical laboratories, including increased urgency, clinical importance, and breadth of application in molecular oncology, as well as more integration of genetic tests into synoptic reporting. Laboratories need to add NGT-based protocols while still providing old tests, and the pace of change is increasing.What follows is one viewpoint on the major challenges in adopting NGTs into diagnostic molecular oncology service.

Choosing a Platform

Instrument selection is a critical decision that has to align with intended test applications, sequencing chemistries, and analytical software. Although multiple platforms are available, a mainstream standard has not emerged. Depending on their goals, laboratories might set up NGTs for improved accuracy of mutation detection, massively higher sequencing capacity per test, massively more targets combined in one test (multiplexing), greater range in sequencing read length, much lower cost per base pair assessed, and economy of specimen volume.

When high-throughput instruments first made their appearance, laboratories paid more attention to the accuracy of base-reading: Less accurate sequencing meant more data cleaning and resequencing (1). Now, new instrument designs have narrowed the differences, and test chemistry can have a comparatively large impact on analytical accuracy (Figure 1). The robustness of technical performance can also vary significantly depending upon specimen type. For example, LifeTechnologies’ sequencing platforms appear to be comparatively more tolerant of low DNA quality and concentration, which is an important consideration for fixed and processed tissues.


Figure 1 Comparison of Sequencing Chemistries

Sequence pile-ups of the same target sequence (2 large genes), all performed on the same analytical instrument. Results from 4 different chemistries, as designed and supplied by reagent manufacturers prior to optimization in the laboratory. Red lines represent limits of exons. Height of blue columns proportional to depth of coverage. In this case, the intent of the test design was to provide high depth of coverage so that reflex Sanger sequencing would not be necessary. Courtesy B. Sadikovic, U. of Western Ontario.


In addition, batching, robotics, workload volume patterns, maintenance contracts, software licenses, and platform lifetime affect the cost per analyte and per specimen considerably. Royalties and reagent contracts also factor into the cost of operating NGT: In some applications, fees for intellectual property can represent more than 50% of the bench cost of performing a given test, and increase substantially without warning.

Laboratories must also deal with the problem of obsolescence. Investing in a new platform brings the angst of knowing that better machines and chemistries are just around the corner. Laboratories are buying bigger pieces of equipment with shorter service lives. Before NGTs, major instruments could confidently be expected to remain current for at least 6 to 8 years. Now, a major instrument is obsolete much sooner, often within 2 to 3 years. This means that keeping it in service might cost more than investing in a new platform. Lease-purchase arrangements help mitigate year-to-year fluctuations in capital equipment costs, and maximize the value of old equipment at resale.

One Size Still Does Not Fit All

Laboratories face numerous technical considerations to optimize sequencing protocols, but the test has to be matched to the performance criteria needed for the clinical indication (2). For example, measuring response to treatment depends first upon the diagnostic recognition of mutation(s) in the tumor clone; the marker(s) then have to be quantifiable and indicative of tumor volume throughout the course of disease (Table 1).

As a result, diagnostic tests need to cover many different potential mutations, yet accurately identify any clinically relevant mutations actually present. On the other hand, tests for residual disease need to provide standardized, sensitive, and accurate quantification of a selected marker mutation against the normal background. A diagnostic panel might need 1% to 3% sensitivity across many different mutations. But quantifying early response to induction—and later assessment of minimal residual disease—needs a test that is reliably accurate to the 10-4 or 10-5 range for a specific analyte.

Covering all types of mutations in one diagnostic test is not yet possible. For example, subtyping of acute myeloid leukemia is both old school (karyotype, fluorescent in situ hybridization, and/or PCR-based or array-based testing for fusion rearrangements, deletions, and segmental gains) and new school (NGT-based panel testing for molecular mutations).

Chemistries that cover both structural variants and copy number variants are not yet in general use, but the advantages of NGTs compared to traditional methods are becoming clearer, such as in colorectal cancer (3). Researchers are also using cell-free DNA (cfDNA) to quantify residual disease and detect resistance mutations (4). Once a clinically significant clone is identified, enrichment techniques help enable extremely sensitive quantification of residual disease (5).

Validation and Quality Assurance

Beyond choosing a platform, two distinct challenges arise in bringing NGTs into the lab. The first is assembling the resources for validation and quality assurance. The second is keeping tests up-to-date as new analytes are needed. Even if a given test chemistry has the flexibility to add analytes without revalidating the entire panel, keeping up with clinical advances is a constant priority.

Due to their throughput and multiplexing capacities, NGT platforms typically require considerable upfront investment to adopt, and training staff to perform testing takes even more time. Proper validation is harder to document: Assembling positive controls, documenting test performance criteria, developing quality assurance protocols, and conducting proficiency testing are all demanding. Labs meet these challenges in different ways. Laboratory-developed tests (LDTs) allow self-determined choice in design, innovation, and control of the test protocol, but can be very expensive to set up.

Food and Drug Administration (FDA)-approved methods are attractive but not always an option. More FDA-approved methods will be marketed, but FDA approval itself brings other trade-offs. There is a cost premium compared to LDTs, and the test methodologies are locked down and not modifiable. This is particularly frustrating for NGTs, which have the specific attraction of extensive multiplexing capacity and accommodating new analytes.

IT and the Evolution of Molecular Oncology Reporting Standards

The options for information technology (IT) pipelines for NGTs are improving rapidly. At the same time, recent studies still show significant inconsistencies and lack of reproducibility when it comes to interpreting variants in array comparative genomic hybridization, panel testing, tumor expression profiling, and tumor genome sequencing. It can be difficult to duplicate published performances in clinical studies because of a lack of sufficient information about the protocol (chemistry) and software. Building bioinformatics capacity is a key requirement, yet skilled people are in short supply and the qualifications needed to work as a bioinformatician in a clinical service are not yet clearly defined.

Tumor biology brings another level of complexity. Bioinformatic analysis must distinguish tumor-specific­ variants from genomic variants. Sequencing of paired normal tissue is often performed as a control, but virtual normal controls may have intriguing advantages (6). One of the biggest challenges is to reproducibly interpret the clinical significance of interactions between different mutations, even with commonly known, well-defined mutations (7). For multiple analyte panels, such as predictive testing for breast cancer, only the performance of the whole panel in a population of patients can be compared; individual patients may be scored into different risk categories by different tests, all for the same test indication.

In large scale sequencing of tumor genomes, which types of mutations are most informative in detecting, quantifying, and predicting the behavior of the tumor over time? The amount and complexity of mutation varies considerably across different tumor types, and while some mutations are more common, stable, and clinically informative than others, the utility of a given tumor marker varies in different clinical situations. And, for a given tumor, treatment effect and metastasis leads to retesting for changes in drug sensitivities.

These complexities mean that IT must be designed into the process from the beginning. Like robotics, IT represents a major ancillary decision. One approach many labs choose is licensed technologies with shared databases that are updated in real time. These are attractive, despite their cost and licensing fees. New tests that incorporate proprietary IT with NGT platforms link the genetic signatures of tumors to clinically significant considerations like tumor classification, recommended methodologies for monitoring response, predicted drug sensitivities, eligible clinical trials, and prognostic classifications. In-house development of such solutions will be difficult, so licensing platforms from commercial partners is more likely to be the norm.

The Commercial Value of Health Records and Test Data

The future of cancer management likely rests on large-scale databases that link hereditary and somatic tumor testing with clinical outcomes. Multiple centers have such large studies underway, and data extraction and analysis is providing increasingly refined interpretations of clinical significance.

Extracting health outcomes to correlate with molecular test results is commercially valuable, as the pharmaceutical, insurance, and healthcare sectors focus on companion diagnostics, precision medicine, and evidence-based health technology assessment. Laboratories that can develop tests based on large-scale integration of test results to clinical utility will have an advantage.

NGTs do offer opportunities for net reductions in the cost of healthcare. But the lag between availability of a test and peer-evaluated demon­stration of clinical utility can be considerable. Technical developments arise faster than evidence of clinical utility. For example, immuno­histochemistry, estrogen receptor/progesterone receptor status, HER2/neu, and histology are still the major pathological criteria for prognostic evaluation of breast cancer at diagnosis, even though multiple analyte tumor profiling has been described for more than 15 years. Healthcare systems need a more concerted assessment of clinical utility if they are to take advantage of the promises of NGTs in cancer care.

Disruptive Advances

Without a doubt, “disruptive” is an appropriate buzzword in molecular oncology, and new technical advances are about to change how, where, and for whom testing is performed.

• Predictive Testing

Besides cost per analyte, one of the drivers for taking up new technologies is that they enable multiplexing many more analytes with less biopsy material. Single-analyte sequential testing for epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase, and other targets on small biopsies is not sustainable when many more analytes are needed, and even now, a significant proportion of test requests cannot be completed due to lack of suitable biopsy material. Large panels incorporating all the mutations needed to cover multiple tumor types are replacing individual tests in companion diagnostics.

• Cell-Free Tumor DNA

Challenges of cfDNA include standardizing the collection and processing methodologies, timing sampling to minimize the effect of therapeutic toxicity on analytical accuracy, and identifying the most informative sample (DNA, RNA, or protein). But for more and more tumor types, it will be possible to differentiate benign versus malignant lesions, perform molecular subtyping, predict response, monitor treatment, or screen for early detection—all without a surgical biopsy.

cfDNA technologies can also be integrated into core laboratory instrumentation. For example, blood-based EGFR analysis for lung cancer is being developed on the Roche cobas 4800 platform, which will be a significant change from the current standard of testing based upon single tests of DNA extracted from formalin-fixed, paraffin-embedded sections selected by a pathologist (8).

• Whole Genome and Whole Exome Sequencing

Whole genome and whole exome tumor sequencing approaches provide a wealth of biologically important information, and will replace individual or multiple gene test panels as the technical cost of sequencing declines and interpretive accuracy improves (9). Laboratories can apply informatics selectively or broadly to extract much more information at relatively little increase in cost, and the interpretation of individual analytes will be improved by the context of the whole sequence.

• Minimal Residual Disease Testing

Massive resequencing and enrichment techniques can be used to detect minimal residual disease, and will provide an alternative to flow cytometry as costs decline. The challenge is to develop robust analytical platforms that can reliably produce results in a high proportion of patients with a given tumor type, despite using post-treatment specimens with therapy-induced degradation, and a very low proportion of target (tumor) sequence to benign background sequence.

The tumor markers should remain informative for the burden of disease despite clonal evolution over the course of multiple samples taken during progression of the clinical course and treatment. Quantification needs to be accurate and sensitive down to the 10-5 range, and cost competitive with flow cytometry.

• Point-of-Care Test Methodologies

Small, rapid, cheap, and single use point-of-care (POC) sequencing devices are coming. Some can multiplex with analytical times as short as 20 minutes. Accurate and timely testing will be possible in places like pharmacies, oncology clinics, patient service centers, and outreach programs. Whether physicians will trust and act on POC results alone, or will require confirmation by traditional laboratory-based testing, remains to be seen. However, in the simplest type of application, such as a patient known to have a particular mutation, the advantages of POC-based testing to quantify residual tumor burden are clear.


Molecular oncology is moving rapidly from an esoteric niche of diagnostics to a mainstream, required component of integrated clinical laboratory services. While NGTs are markedly reducing the cost per analyte and per specimen, and will certainly broaden the scope and volume of testing performed, the resources required to choose, install, and validate these new technologies are daunting for smaller labs. More rapid obsolescence and increased regulatory scrutiny for LDTs also present significant challenges. Aligning test capacity with approved clinical indications will require careful and constant attention to ensure competitiveness.


1. Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012; doi:10.1155/2012/251364.

2. Brownstein CA, Beggs AH, Homer N, et al. An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge. Genome Biol 2014;15:R53.

3. Haley L, Tseng LH, Zheng G, et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal ­cancers. [Epub ahead of print] Modern Pathol July 31, 2015 as doi:10.1038/modpathol.2015.86.

4. Butler TM, Johnson-Camacho K, Peto M, et al. Exome sequencing of cell-free DNA from metastatic cancer patients identifies clinically actionable mutations distinct from primary ­disease. PLoS One 2015;10:e0136407.

5. Castellanos-Rizaldos E, Milbury CA, Guha M, et al. COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing. Methods Mol Biol 2014;1102:623–39.

6. Hiltemann S, Jenster G, Trapman J, et al. Discriminating somatic and germline mutations in tumor DNA samples without matching normals. Genome Res 2015;25:1382–90.

7. Lammers PE, Lovly CM, Horn L. A patient with metastatic lung adenocarcinoma harboring concurrent EGFR L858R, EGFR germline T790M, and PIK3CA mutations: The challenge of interpreting results of comprehensive mutational testing in lung cancer. J Natl Compr Canc Netw 2015;12:6–11.

8. Weber B, Meldgaard P, Hager H, et al. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays. BMC Cancer 2014;14:294.

9. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013;339:1546–58.

10. Heitzer E, Auer M, Gasch C, et al. Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. Cancer Res 2013;73:2965–75.

11. Healy B. BRCA genes — Bookmaking, fortunetelling, and medical care. N Engl J Med 1997;336:1448–9.




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New methods for Study of Cellular Replication, Growth, and Regulation

Writer and Curator: Larry H Bernstein, MD, FCAP


This article is the first in a series on genomics, epigenomics and cancer.  It is necessary here to introduce the large advancement in the technological advances that have followed the Human Genome Project and its thrust into the domain of genome expression.  While the genome is the code that is passed on from generation to generation in a family chain, and while there now is an ability to trace genes that have existed and are traceable by evolutionary history to early eukaryotic species, that portion of the cell line is defining and only modified in time.  It is only a beginning in the unraveling of the question – What is life?

This article has the following structure:

1.1.1       Gene Amplification

1.1.2       Protein-binding Receptors

1.1.3 Advanced Proteomic Technologies for Cancer Biomarker Discoveries State of the art technologies 2D difference gel electrophoresis (2DIGE) MALDI imaging technology (see also 1.1.5) Electron Transfer Dissociation Reverse-phase Protein Array (RPA) Principles of Protein Microarrays Disposable reagentless electrochemical immunosensor array based on a polymer/sol/gel membrane for simultaneous measurement of several tumor markers

1.1.4 p16INK4a Expression Correlates with Degree of Cervical Neoplasia: A Comparison with Ki-67 Expression and Detection of High-Risk HPV Types

1.1.5 Quantitative real-time detection of magnetic nanoparticles by their nonlinear magnetization

1.1.6 Proteomics and biomarkers Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine Multiplexed proteomic analysis of oxidation and concentrations of CSF proteins in Alzheimer’s disease The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients Determination of non-α1-antichymotrypsin-complexed PSA as an indirect measurement of free PSA: analytical performance and diagnostic accuracy Ultrasensitive densitometry detection of cytokines with nanoparticle-modified aptamers Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker

1.1.7 Mass Spectrometry Methods LC-MS/MS quantification of Zn-α2 glycoprotein: A potential serum biomarker for prostate cancer A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples Mass Spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications Current state and future directions of neurochemical biomarkers for Alzheimer’s disease Use of SELDI-TOF mass spectrometry for identification of new biomarkers: potential and limitations

1.1.1 Gene Amplification

An increase in the number of copies of a gene. There may also be an increase in the RNA and protein made from that gene. Gene amplification is common in cancer cells, and some amplified genes may cause cancer cells to grow or become resistant to anticancer drugs. Genes may also be amplified in the laboratory for research purposes.


Actinin-4 gene Amplification in Ovarian Cancer: A Candidate Oncogene Associated with Poor Patient Prognosis and Tumor Chemoresistance

Yamamoto S, Tsuda H, Kazufumi H, Onozato K, …, Matsubar O

Medscape 6/18/2009


Actinin-4, an isoform of non-muscular α-actinin, enhances cell motility by bundling the actin cytoskeleton. We previously reported a prognostic implication of high histochemical expression of actinin-4 protein in ovarian cancers.  Chromosomal gain or amplification of the 19q 12-q13 region has been reported in ovarian cancer. We hypothesized that the actinin-4 (ACTN4) gene might be a target of the 19q 12-q13 amplicon and play an essential role in ovarian cancer progression. In total, we investigated 136 advanced-stage ovarian cancers the copy number of the ACTN4 gene on chromosome 19q3, and used fluorescence in situ hybridization to determine the correlation of the ACTN4 copy number with actinin-4 protein immunoreactivity and major clinicopathological factors. We detected a higher copy number ( > 4) of  of the ACTN4 gene in 29 (21%) cases and it was associated with the intensity of the actinin-4 immunoreactivity (p < 0.0001), a high histological tumor grade (p < 0.030), a clear-cell adenocarcinoma histology (p = 0.012), resistancxe to first- line chemotherapies (p = 0.028), and poor patient outcome (p=0.0011). Uni-
variate analyses using the Cox regression model showed that a higher ACTN4 gene copy numberwas predicted patient outcome more accurately than high actinin-4 immunoreactivity (relative risk: 2.48 vs 1.55). Multivariate analysis indicated that a higher copy number of the ACTN4 gene may be a targetof the 19q amplicon, acting as a candidate oncogene, and serve as a predictor of poor outcome and tumor chemoresistance in patients with advanced-stage ovarian cancers (from Modern Pathology).

1.1.2       Protein-binding Receptors

Customizing the Targeting of IGF-1 Receptor

Renato Baserga
Medscape 5/6/2009. From Future Oncology

The type 1 IGF receptor (IGF—IR) is activated by two ligands, IGF-1 and IGF-2, and by insulin at supraphysiological concentrations. It plays a significant role in the growth of normal and abnormal cells, and antibodies against the IGF-IR are now in clinical trials. Targeting of the IGF-IR in cancer cells (by antibodies or other means) can be improved by the appropriate selection of responsive tumors.

The prominence of IGF-IR has increased considerably in the past few years, progressing from a redundant insulin receptor to one that is important in cell and body growth, cell survival and malignant transformation. The IGF-IR can send either a mitogenic or a differentiation signal depending on substrate availability. In many cell types (fibroblasts, epithelial cells, etc.) the IGF-IR sends an unambiguous mitogenic, antiapoptotic signal. In other cell types, such as myeloid cells, neuronal cells and others, activation of IGF-IR induces differentiation. When cells do not express or express very low levels of IRS-1, a substrate of both the IGF_IR and the insulin receptor (InR), IGF-1 induces differentiation. This is the case with 32D myeloid precursor cells that do’t express IRS-1 and are induced to differentiate by IGF-1. Ectopic expression of IRS-1 in 32D cells abrogates differentiation; the cells become transformed and even form tumors in mice. IRS-1 activates the P13K pathway, which is the main mitogenic pathway originating from the IGF-IR. However, the shc-Ras-ERKs pathway also plays a role in the mitogenic signal of the IGF-IR, with the two converging at GSK3-β. The demonstration that knockout mouse embryo cells for the IGF-IR receptor were refractory to transformation by viruses, oncogenes and overexpressed growth factor receptors clearly demonstrated the major role played by the IGF-IR in cellular transformation.

Methods for Targeting the IGF-1 Receptor

·        The original methods for targeting the IGF-1 receptor in experimental animals were antisense strategies and dominant-negative mutants of the receptor. They are obsolete.
·        Several antibodies to the IGF-IR are effective in inhibiting tumor growth in vitro and in mice. They are now in Phase I clinical trials.
·        Other investigators have identified specific inhibitors of the IGF-1 receptor tyrosine kinase activity. (cyclolignans)
·        To induce apoptosis, it is probably necessary to downregulate the receptor. Without downregulation, there is inhibition of growth, but no apoptosis.
·        Targeting the ligands gives good results in mice, but fails in humans. Adult mice express only IGF-1, but humans keep synthesizing both IGF-1 and IGF-2 in adult life.

Summary of IRS-1

·        Insulin receptor substrate (IRS-1) is a multitask protein that interacts with many other proteins.·        IRS-1 is mitogenic, inhibits differentiation, protects from apoptosis and regulates cell (and body size).·        IRS-1 is essential to mitogenic IGF-IR signaling.

·        IRS-1 is activated by the EGF receptor, cMet and the Ewing’s sarcoma oncogene.

·        IRS-1 plays a significant role in transformation by T antigen and v-src.

·        Doenregulation of IRS-1 causes growth arrest and differentiation.

·        Nuclear IRS-1 acts as a transcriptional cofactor for both RNA pol 1 and 2-directed genes.

·        IRS-1 effects on cells can be dissociated from the effects of IGF-IR, IRS-2 and insulin receptor.

·        IRS-1 is a biomarker of sensitivity of cancer cells to IGF-IR targeting.

·        Hypothesis: IRS-1 is an antitumor suppressor, similar to anti-p53 protein.


The ability of IRS-1 to cause cell transformation, and the tendency to lose the transformed phenotype in cells in which IRS-1 is low or has been downregulated, suggests that the importance of the IGF-IR in cancer may be dependent on IRS-1 as much as on the receptor itself. When IRS-1 is activated directly, for instance by v-src, the IGF-IR is no longer a requiremeny for malignant transformation.  Metastases are very susceptible to IGF-IR therapy.

IGF-IR Targeting Summary

  • In the absence of IRS-1, the IGF1R sends a differentiation signal, which becomes mitogenic with IRS1 expression, and targeting IGF1R in cells that do not express IRS-1 may be counterproductive.
  • In colon cancer liver metastases the cancer cells of awash in IGF-1.
  • In Ewing’s sarcoma, a tumor sensitive to IGF1r targeting in clinical trials, there is an autocrine mechanism that may make the cancer cells IGF-1 dependent, but an oncogene/IRS-1 interaction may also make these cells incapable of switching to other growth factors.
  • IGF-1R sends a potent anti-apoptotic signal, independent of its mitogenicity. This property could be exploited to increase chemo- or radio- toxicity.
  • IGF-1R expression is required for anchorage independence.

1.1.3 Advanced Proteomic Technologies for Cancer Biomarker Discoveries State of the art technologies

Wong SCC, Chan CML, Ma BBY,…,Chan ATC.

Medscape 6/10/2009. From Expert Review of Proteomics.

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. The topic of discussion is four state of the art technologies: 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array.  These have contributed to large advances in proteomic technologies from 1997-2008. 2D difference gel electrophoresis (2DIGE)

The 2D DIGE method is an improved 2GE technique. Two different protein samples (e.g., control and disease) and, optionally, one reference sample (e.g., control and disease together) are labeled with one of three different fluorophore: cyanine (cy)2, 3 or 5. These fluorophores have the same charge, similar molecular weight and distinct fluorescent properties, allowing their discrimination during scanning using appropriate optical filters.Two types of cyanine dyes are available: CyDyeTM  DIGE Fluor minimal dyse and CyDye DIGE Fluor saturation dye (GE Healthcare, Uppsala, Sweden).The minimal dye labelks a small percentage of lysine residues with minimal change in the electrophoretic mobility pattern of the protein, whereas the saturation dye labels all available cysteine residues and is, therefore, more sensitive, but causes EP mobility shift of labelled proteins.  Different types of protein sample may be used.Labeled sample pairs are mixed and  run in a single gel.The same pooled reference sample is used for all gels within an experiment.The gel is scanned at three wavelengths for Cy2 (488 nm), Cy3 (532 nm) and Cy5 (633 nm), and a gel image for each of the samples is obtained.Variation between the gels is minimized. Correct matching of protein spots is improved.Normalization and quantitation of the spots is most accurate.The linear dynamic range is four orders of magnitude and it is fully compatible for quantitation with MS. The technique is mainly used for the discovery of novel biomarkers. MALDI imaging technology (see also 1.1.5)

MALDI Imaging Mass Spectrometry gives a deeper understanding of biochemical processes in the tumor cells and tissues. Immunohistochemistry is limitated, but the MALDI technique is high-throughput.MALDI IMS was developed to allow researchers to analyze proteomic expression profiles directly from patient tissue sections.The tissue is first mounted, then MALDI matrix is applied onto the tissue sample and MALDI MS is applied to obtain mass spectra from predefined locations across the tissue section.All acquired spectra are then compiled into a composite 2D map for the tissue sample.The expression profiles of numerous proteins can be obtained without the need for antibodies. It is also possible to correlate the mapping with tissue histology.

Post-translational modifications have a role in structure and function of proteins: protein folding, protein localization, regulation of activity and mediation of protein-protein interaction. Two common forms of PTM have been implicated in cancer neoplasia: phosphorylation and glycosylation.  Phosphoproteomic studies led to identification of novel tyrosine kinase substrates in breast cancer, and to discovery of novel therapeutic targets for brain cancer, and to increased understanding of signaling pathways in lung cancer.  The identification of novel therapeutic targets for ovarian cancer resulted from identification of abnormally glycosylated proteins – mucins. Electron Transfer Dissociation

Electron Transfer Dissociation is a recently developed technique for the analysis of peptides by MS, utilizing radiofrequency quadrupole ion traps such as 2D linear IT, spherical IT and OrbitrapTM (Thermo Fisher,MA).Peptides are fragmented by transfer of electrons from anions to induce cleavage of CαN bonds along the peptide backbone, producing c- and z-type ions. In contrast to CID, ETD preserves the localization of labile PTM and provides peptide-sequence information, but it fails to fragment peptide bonds adjacent to proline.CID and ETD should be used to complement each other. An advantage of the TED is that in the analysis of phosphopeptides a near complete series of c- and z-ions is observed without the loss of phosphoric acid. The method has provided for proteomic researchers a tool for comprehensive analysis of peptides and their PTMs. Reverse-phase Protein Array (RPA)

Then there is the Reverse-phase Protein array, which has the advantage that it identifies changes associated with the development of cancer. The identification of such proteins can be used as biomarkers for diagnosis, prognosis, treatment decisions and therapeutic monitoring. Still, patient samples pose a challenge:

  • Proteomic patterns differ among cell types;
  • Protein expression changes dynamically over time;
  • Proteins have a broad dynamic range of expression levels spanning several orders of magnitude;
  • Proteins can be present in multiple forms, such as polymorphysms and splice variants;
  • Traditional proteomic methods, such as, @DE, require larger amounts of protein than those obtained from biopsy samples;
  • Many existing proteomic technologies cannot ber used to study protein-protein interactions.

The method of RPA is simple and requires the spotting of patient samples in an array format onto a nitrocellulose support.Each array is incubated with a particular antibody, and signal intensity is proportional to the amount of analyte. Signal detection is by fluorescence, chemiluminescence or colorimetric methods.  The results are qwuantified by scanning and analyzed by softwares such as P-SCAN and ProteinScan.

Main advantages of RPA are:

  • Various types of biological samples;
  • Investigation of PTMs;
  • Protein-protein interactions;
  • Labeling of samples with fluorescent dyes or mass tags;
  • Quantitation within the linear range of detection;
  • Direct measurement of target proteins by spotting reference standards.

Key Issues

  • 2DE couple with MS has been a mainstay for discovery of novel biomarkewrs;
  • 2D DIGE has improved quantification accuracy;
  • MALDI imaging MS allows detedtion and comparison with histopathology;
  • ETD-MS has opened up the possibility of identifying the structure and localization of PTM and the peptide/protein.
  • RPA is a powerful tool for high-throughput validation across hundreds of samples.  Principles of Protein Microarrays

Preface, Foreward and Chapter 1: In Protein Microarrays, Ed. Mark Schena
Mark Schena, Joseph L. Hackett and Emanuel F. Petricoin
Jones and Bartlett Publ. 2002, ON, CA

What is true inside the cell cannot always be recapitulated outside the cell.  The year is 1986 and the second year of graduate school of UCSF. With cloned receptor in hand (just isolated by Roger Miesfeld), I set out to test whether glucocorticoid receptor function could be recapitulated in yeast cells. This might allow us to test evolutionary nconservation in eukaryotes.  Remarkably, the rat receptor sprsang to life on the first attempt, producing a diagnostic blue colot change in yeast cells expressing a β-galactosidase fusion and a broad smile on the face of a young scientist. Receptor experiments in yeast necessarily required grinding up yeast cells, fractionating the proteins by denaturing polyacrylamide gel electrophoresis, transferring the proteins onto nitrocellulose, probing the immobilized proteins with a monoclonal antibody, and examining the filter to confirm the presence of the expressed rat protein.

Protein-ntibody interactions on protein chips are determined by complex associations between epitopes on the target protein and the antigen-binding site on the detection molecule. Individual protein-ligand pairs can possess widely different affinities.  Proteomic microarrays require capture and detection molecules with high affinities and low dissociation rates . For these and other reasons protein chips are more challenging than DNA chips. Antibodies, aptamers, recombinant proteins, peptides, phage, evem small molecular weight chemicals/drugs can be used as a bait molecule and/or detection reagent. The molecule may be an antibody or the cellular lysate itself, which are immobilized onto the substratum and act as a bait molecule.  Each spot contains one type of immobilized antibody or bait protein. The first problem is the vast range of concentrations to be detected (up to a factor of 1010 .  Adequate sensitivity must be achieved (at least femtomolar range), and the amplification chemistry must be tolerant to the large dynamic range of the analytes.

Microarrays are analytical devices that possess four distinct characteristics:

  1. Microscopic target elements or spot;
  2. Planar substrates;
  3. Rows and columns of elements; and
  4. Specific binding between microarray target elements on the substrate and probe molecules in solution.

The scope of microarray research includes:

  1. Gene expression
  2. Signal transduction
  3. Genome mismatch scanning
  4. Inflammation
  5. Cancer
  6. Cell cycle
  7. DNA replication
  8. Oxidative stress
  9. Hormone action
  10. Apoptosis
  11. Neurodegenerative disease
  12. Infectious disease
  13. Cytoskeleton, and
  14. Protein trafficking.

The proliferation of microarrays beyond the realm of DNA and gene expression was inevitable, and the idea of making and using microarrays of proteins, lipids, carbohydrates, and small molecules was an obvious extension of the original DNA microarray format. This exciting technology area provides the foundation for the book, Protein Microarrays.  Proteins, not mRNAs are the true functional components of cells. They mediate gene regulation, enzyme catalysis, cellular metabolism, DNA replication, and cell division and confer cell shape and mobility and the capacity to communicate within and between cells. a hypothetical 400 amino acid protein would have a molecular weight of 54 kDa.
Many cellular proteins fall in the molecular weight range of 10-125 kDa, and nearly every human protein weighs < 500 kDa.

The 20 amino acids are chemically diverse and correspondingly confer to the proteins their structural and functional diversity and impart their catalytic specificity and binding properties. The amino acids are bound in the protein by the amino acid side chains of the polypeptide. The nh-CO peptide unit has a partial double-bond character due to the amide bond, and its conformations are restricted by that structure. In addition, proteins have secondary, tertiary and quaternary structure. Hydrophobic amino acids are in the interior, and hydrophilic amino acid residues are on the exterior. The hydrophilic exterior allows for water solubility.

The following are microarray assay formats used in expression profiling:

  1. Protein expression
  2. Serum-based diagnostics
  3. Protein-protein binding
  4. Drug-target binding
  5. Receptor-epitope binding.  Disposable reagentless electrochemical immunosensor array based on a polymer/sol/gel membrane for simultaneous measurement of several tumor markers

Wu J, Yan F, Zhang X, Yan Y, Tang J, Ju H.
Clin Chem 2008; 54(9):1481-1489.

Background: A reagentless sensor array for simultaneous multianalyte testing (SMAT) may enable accurate diagnosis and be applicable for point-of-care testing. We developed a disposable reagentless immunosensor array for simple immunoassay of panels of tumor markers. Methods: We carried out SMAT with a direct capture format, in which colloidal gold nanoparticles with bound horseradish peroxidase (HRP)-labeled antibodies were immobilized on screen-printed carbon electrodes with biopolymer/sol-gel to trap their corresponding antigens from sample solution. Upon formation of immunocomplex, the direct electrochemical signal of the HRP decreased owing to increasing spatial blocking, and the analytes could be simultaneously determined by monitoring the signal changes.
Results: The proposed reagentless immunosensor array allowed simultaneous detection of carcinoma antigen 153, carcinoma antigen 125, carbohydrate antigen 199, and carcinoembryonic antigen in clinical serum samples in the ranges of 0.4–140 kU/L, 0.5–330 kU/L, 0.8–190 kU/L, and 0.1–44 μg/L, respectively, with detection limits of 0.2 kU/L, 0.5 kU/L, 0.3 kU/L, and 0.1 μg/L corresponding to the signals 3 SD above the mean of a zero standard. The interassay imprecision of the arrays was <9.5%, and they were stable for 35 days. The positivity detection rate of panels of tumor markers was >95.5% for 95 cases of cancer-positive sera. Conclusions: The immunosensor array provides a SMAT with short analytical time, small sampling volume, no need for substrate, and, no between-electrode cross-talk. This method not only proved the capability of the array in point-of-care testing, but also allowed simultaneous testing of several tumor markers.

Cancer is one of the leading causes of mortality, and early clinical diagnosis is crucial for successful treatment of the disease. Many immunosensors and immunoassay methods have been developed for the determination of a single tumor marker, whose concentration in human serum is associated with the stages of tumors (1)(2)(3)(4). Because many cancers express 1 marker [e.g., breast cancer is associated with carcinoma antigens 153 and 125 (CA 153 and CA 125)1 and carcinoembryonic antigen (CEA)], and concentrations of several tumor markers often increase in the serum of a patient, accurate simultaneous multianalyte test (SMAT) of combinations of tumor markers may improve the diagnosis of certain types of tumor (5)(6)(7)(8).

SMAT may offer a shorter analytical time, higher sample throughput, lower sampling volume, and lower cost per assay compared with traditional single-analyte tests (9)(10). Thus, multilabel assays and spatially resolved assay systems have been developed as the main modes to perform SMAT (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Application of the multilabel assays has been limited by difficulty in accurate quantification due to different optimal assay conditions and the signal overlap of different labels (11)(12)(13). Although a set of substrate zone-resolved techniques have been proposed to overcome these drawbacks (14)(15), the restriction in the number of available labels still greatly limits their application.

Spatially resolved assays with a single label seem well suited for performing SMAT, and optical SMAT, relying on fluorescence emission and optical reflectance, has been developed into mature technology. Optical SMAT, however, often needs an expensive array detector, such as a charge-coupled device camera (16)(17). Electrochemical array, which is distinguished by its convenient miniaturization for high-throughput systems, low assay cost, and absence of sophisticated and expensive array detectors, shows promising application in cancer screening (18)(19). Electrochemical cross-talk caused by diffusion of the detectable enzymatic products is the main problem in the fabrication of electrochemical array. To solve this problem, many approaches have been followed. One approach, for example, is to ensure that the distance between adjacent electrodes is larger than the diffusion distance of enzymatic product (20)(21)(22)(23), but such an approach conflicts with the goal of miniaturization. Another simple method to completely avoid the electrochemical cross-talk can be achieved by immobilizing the electron-transfer mediator on an individual immunosensor to shuttle electrons (24)(25), but this approach requires the addition of hydrogen peroxide, leading to limited practical application.

A reagentless electrochemical immunosensor is an attractive strategy (26). In our previous work, we prepared several reagentless immunosensors by using sol-gel matrix to immobilize immunoreagents and detected the direct electron transfer of labeled enzyme, horseradish peroxidase (HRP) (27)(28)(29)(30)(31). To achieve SMAT, this work further fabricated a reagentless immunosensor array by individually embedding 4 kinds of HRP-labeled antibody-modified gold nanoparticles in a newly designed biopolymer/sol-gel matrix formed on screen-printed carbon electrodes (SPCEs), where the HRP-Ab-Au nanoparticles were limited in the holes of the biopolymer/sol-gel film. Chitosan, a biopolymer with excellent film-forming ability, biocompatibility, nontoxicity, and high mechanical strength, acted as the adhesion frame in the synthesis of the sol-gel and made the electrical communication between redox sites of the enzyme and sensing surface easier due to the cooperative effort of chitosan and sol-gel matrix. The presence of gold nanoparticles accelerated the electron transfer between immobilized HRP and the electrode and increased the hole size for improving the permeability of the sol-gel matrix so that the antigens in solution could easily penetrate into the sol-gel film for immunoreaction. Upon formation of immunocomplexes, the electrochemical responses decreased due to increasing spatial blocking, leading to the reagentless immunosensing to corresponding antigens without cross-talk. The proposed electrochemical immunosensor array had high analyte throughput, showed acceptable comparability to conventional methods for measuring several tumor markers, could be fabricated with mass production techniques, and thus provided the potential for application in point-of-care testing (POCT).

Schematic diagrams of immunosensors array and multianalyte electrochemical immunoassay system.

Schematic diagrams of immunosensors array and multianalyte electrochemical immunoassay system.

Figure 1.

Schematic diagrams of immunosensors array and multianalyte electrochemical immunoassay system.

(a), Nylon sheet, (b), silver ink, (c), graphite auxiliary electrode, (d), Ag/AgCl reference electrode, (e), graphite working electrode, (f), insulating dielectric.

The DPV curves of both the bare and biopolymer/sol-gel modified SPCEs in 0.2 mol/L PBS, pH 6.9, did not show any detectable signal in the applied potential window (Fig. 3 ). After we embedded 0.17 μL of 50 mg/L HRP-anti-CA 153 in the biopolymer/sol-gel, the modified SPCEs displayed a sensitive peak around −540 mV (vs Ag/AgCl) (curve d, Fig. 3 ), which was close to the reduction peak potential of HRP/biopolymer/sol-gel prepared with 0.17 μL of 2.0 mg/L HRP (curve c, Fig. 3 ), indicating the direct electron transfer between electrode and the labeled HRP with regard to Fe(III)-Fe(II) conversion. The small difference of peak potentials between HRP-anti-CA 153 and HRP resulted from the change of microenvironment around HRP molecules because of the presence of antibody. In the presence of gold nanoparticles in the biopolymer/sol-gel, the reduction peak of the equal amount of HRP-antibody conjugate increased 2.1-fold (curve e, Fig. 3 ). The cyclic voltammetric experiments at different gold electrodes showed the same appearance, and upon incorporation of gold nanoparticles into the biopolymer/sol-gel at SPCEs, the reduction peak current at the same scan rate increased 1.98-fold (see Supplemental Fig. 1 in the Data Supplement that accompanies the online version of this article at  http://www.clinchem.org/content/vol54/issue9). Thus the Au nanoparticles could accelerate the direct electrochemistry of HRP to further amplify the detectable signal. This peak was also 2.7 times higher than that of HRP-anti-CA 153-Au nanoparticles/sol-gel modified SPCE (curve f, Fig. 3 ), indicating the positive effects of chitosan with good biocompatibility and hydrophilicity (35), which enhanced water uptake and swelling of the film and led to better permeability of the film for the transfer of counter ions to neutralize the charge change during the redox process and a favorable microenvironment for electron hopping or electron self-exchange between immobilized HRP molecules (36). Thus electron transfer kinetics and direct electrochemical signal increased. After the modified SPCE was incubated with CA 153, the direct electrochemical signal decreased markedly due to the increased barrier that resulted from the formation of immunocomplex (curve g, Fig. 3 ), leading to a reagentless immunosensing method for antigen detection.

DPVs of bare SPCE

DPVs of bare SPCE

DPVs of bare SPCE (a), biopolymer/sol-gel (b), HRP/biopolymer/sol-gel (c), HRP-anti-CA 153/biopolymer/sol-gel (d), HRP-anti-CA 153-Au nanoparticles/biopolymer/sol-gel (e), HRP-anti-CA 153-Au nanoparticles/sol-gel modified SPCE in pH 6.9 PBS (f), and panel e in pH 6.9 PBS after incubation in 20 μL of 50 kU/L CA 153 at room temperature for 40 min (g).

The formation of immunocomplex depended on the incubation temperature and time. For the sake of convenient manipulation, the incubation step was performed with 20 μL antigen solution or the mixture of antigens for SMAT at room temperature, after which the DPV response of the labeled HRP decreased with increasing incubation time and reached a relatively stable value at 30–40 min (Fig. 4A ), indicating saturated formation of immunocomplex in the membrane. Thus, 40 min was chosen as the optimal incubation time for SMAT.

Dependences of DPV responses of immunosensors on incubation time

Dependences of DPV responses of immunosensors on incubation time

Dependences of DPV responses of immunosensors on incubation time (A) and pH of detection solution (B) for CA 153, CA 125, CA 199, and CEA.

Table 1.

Positivity detection rates of clinical sera.

Sample n Associated tumor markers Positive cases, n Positivity detection rate, %
Colorectal or gastric cancer 53 CA 199, CEA 531 100
Epithelial ovarian cancer 22 CA 125, CA 199, CEA 211 95.5
Breast cancer 8 CA 153, CA 125, CEA 81 100
Lung cancer 12 CA 199, CEA 121 100
Normal serum 20 CA 153, CA 125, CA 199, CEA 22 10

In comparison with previous reports (24)(25), this array avoids the addition of mediator to shuttle electrons, and thus can exclude the electrochemical cross-talk at the electrode dimensions used here. Furthermore, the measurement of the direct electrochemical signal of HRP labeled to immunoreagents also avoids the need for other reagents in the detection process. Although the measurements show acceptable results, adding sulfite in the detection solution is not the best solution for the removal of oxygen. Thus, a system has been developed for POCT to exclude oxygen from the detection solution (see Supplemental Fig. 3 in the online Data Supplement).  p16INK4a Expression Correlates with Degree of Cervical Neoplasia: A Comparison with Ki-67 Expression and Detection of High-Risk HPV Types

S Nicholas Agoff, Patricia Lin, Janice Morihara, Constance Mao, Nancy B Kiviat and Laura A Koutsky
Mod Pathol 2003;16(7):665–673

Although recent studies have suggested that p16INK4a may be a useful surrogate biomarker of cervical neoplasia, Ki-67 and human papillomavirus testing have also been shown to be useful in detecting neoplasia. To help delineate the utility of p16INK4a, biopsy samples (n = 569: negative, 133; reactive, 75; atypical, 39; low grade, 76; moderate, 80; and severe intraepithelial neoplasia, 113; also, squamous cell carcinoma, 46; adenocarcinoma, 7) were analyzed by immunohistochemistry for expression of p16INK4a and Ki-67 (n = 432), as well as by in situhybridization for human papillomavirus Type 16 (n = 219). Testing for high-risk human papillomavirus types by polymerase chain reaction and HybridCapture2 was performed on concurrent cervical swab specimens. Recuts of the original blocks were reexamined (n = 198). Endometrial biopsies (n = 10) were also analyzed for p16INK4a expression. Degree of p16INK4a and Ki-67 expression correlated with degree of cervical neoplasia (P < .001) and with presence of high-risk human papillomavirus types (P < .001). There was no relationship between p16INK4a overexpression and inflammation or hormonal status. Ki-67 expression correlated with inflammation (P = 0.003) and was expressed in more reactive and atypical lesions than p16INK4a (P = 0.008). Probes for human papillomavirus 16 stained 54% of cervical neoplastic lesions; the degree of staining correlated significantly with degree of neoplasia (P < .001) and p16INK4astaining (P < .001). Interobserver reproducibility was substantial for p16INK4a and Ki-67 interpretation (weighted kappa: 0.74 and 0.70, respectively). Expression of p16INK4a was observed in all endometrial biopsies. Compared with Ki-67 expression and detection of high-risk human papillomavirus, p16INK4a was less likely to be positive in samples from women with negative, reactive, and atypical biopsies. Although expression of p16INK4ain endometrial epithelium may be problematic in terms of screening, the potential of p16INK4a as a screening test warrants investigation.

The screening of women by Pap smear has led to a remarkable decline in the mortality from cervical cancer; however, secondary to subjective criteria, interpretation of Pap smears is subject to marked inter- and intraobserver variability as well as having a relatively low sensitivity for cervical neoplasia on a single sample (as low as 66% sensitivity for biopsy-proven high-grade squamous intraepithelial lesions [HSIL]) (1, 2). Recently, histology, which is thought of as the gold standard for the diagnosis of cervical neoplasia, has also been found to suffer from marked intra- and interobserver variability, and testing for high-risk human papillomavirus (HPV) by Hybrid Capture 2, which has been shown to be very sensitive in the detection of cervical neoplasia and useful in the triaging of ASCUS smears, has a low specificity for cervical neoplasia (1,3). Thus, new biomarkers that are more sensitive and specific in the detection of cervical neoplasia and more reproducible than cervical cytology are needed.

Human papillomaviruses (HPV) are known to be a major causative agent in cervical neoplasia and invasive cervical carcinoma. Many different HPV types associated with cervical neoplasia have been discovered, and they have been subdivided into high- and low-risk categories based on their association with invasive cervical carcinoma (4). This association is based, in part, on the relative affinity that the HPV-type specific oncoproteins E6 and E7 bind to cellular regulatory proteins, specifically, the p53 tumor suppressor protein and the retinoblastoma protein (Rb) (5). Inactivation of these factors, either by degradation (p53) or functional inactivation (Rb), leads to disruption of the cell cycle and increased proliferation, thought to ultimately give rise to carcinoma.

p16INK4a is a cyclin-dependent kinase inhibitor that regulates the activity of cyclin-dependent kinases 4 and 6 and is often inactivated in many cancers by genetic deletion or hypermethylation (6). In non-HPV–associated tumors, this inactivation leads to increased cyclin-dependent kinase activity and inactivation of Rb. However, in HPV-associated tumors, inactivation of Rb by E7 leads to markedly increased levels of p16INK4a. Recent studies have documented overexpression of p16INK4a not only in cervical intraepithelial neoplasia (CIN) but in cervical cancer as well (6, 7, 8, 9, 10).

For p16INK4a, the results were reported in semiquantitative fashion (negative, or 1+ to 3+) based on none, 5–25%, 25–75%, and >75% of cells immunostained in a lesion. Strong nuclear as well as cytoplasmic staining was considered a positive reaction. Wispy weak cytoplasmic staining present in rare cells (<5%) was considered plusminus, and for analysis was grouped into the negative category. For Ki-67, the results were also reported in a semiquantitative fashion as cells in the lower 1/3 of the epithelium staining (i.e., usually basilar cell staining), cells in the middle 1/3–2/3 staining, or cells in the upper 1/3 staining (14). Strong nuclear staining was considered a positive reaction. Stains were analyzed by two authors (SNA and JM) for reproducibility; each was blinded to the other’s result.

The degree of p16INK4a expression correlated well with the degree of cervical neoplasia, and this correlation improved slightly when compared with the recut slide diagnosis (P < .001; Fig. 1; Tables 1 and 2). There was very little expression in negative and reactive lesions, with only 11% to 12% showing greater than or equal to1+ staining (24 of 208 -original diagnosis, 12 of 112 recut diagnosis). 57% of the CIN I cases had greater than or equal to1+ expression, compared with 75% of CIN II lesions and 91% of CIN III lesions. On the recut diagnosis, 97% of CIN III lesions stained greater than or equal to1+. There were 10 (9%) CIN III original diagnosis that did not stain for p16INK4a, but on review, the majority of these were secondary to the lesion being cut through and not present on the immunohistochemistry (IHC) slide. For the recut diagnosis, there was only 1 (3%) case that did not stain with p16INK4a, and on review, two of three pathologists agreed that this represented CIN III, whereas the third felt it represented atypical squamous metaplasia. p16INK4a expression of 1+ or greater was present in 89%(47/53) of the invasive carcinomas. Review of negative cases confirmed the carcinoma diagnosis.

p16INK4a and Ki-67 expression in normal, low-grade squamous dysplasia, and high grade squamous dysplasia

p16INK4a and Ki-67 expression in normal, low-grade squamous dysplasia, and high grade squamous dysplasia

p16INK4a and Ki-67 expression in normal cervical squamous mucosa (A, H&E stain; B, p16INK4a; C, Ki-67), low-grade squamous dysplasia (CIN I; D, H&E stain; E, p16INK4a; F, Ki-67), and high grade squamous dysplasia (CIN III; G, H&E stain; H, p16INK4a, I, Ki-67).  Quantitative real-time detection of magnetic nanoparticles by their nonlinear magnetization

A novel method of highly sensitive quantitative detection of magnetic nanoparticles (MP) in biological tissues and blood system has been realized and tested in real time in vivoexperiments. The detection method is based on nonlinear magnetic properties of MP and the related device can record a very small relative variation of nonlinear magnetic susceptibility up to 108 at room temperature, providing sensitivity of several nanograms of MP in 0.1mlvolume. Real-time quantitative in vivomeasurements of dynamics of MP concentration in blood flow have been performed. A catheter that carried the blood flow of a rat passed through the measuring device. After an MP injection, the quantity of MP in the circulating blood was continuously recorded. The method has also been used to evaluate the MP distribution between rat’s organs. Its sensitivity was compared with detection of the radioactive MP based on isotope of Fe59. The comparison of magnetic and radioactive signals in the rat’s blood and organ samples demonstrated similar sensitivity for both methods. However, the proposed magnetic method is much more convenient as it is safe, less expensive, and provides real-time measurementsin vivo. Moreover, the sensitivity of the method can be further improved by optimization of the device geometry.

1.1.6  Proteomics and biomarkers Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine

Kageyama S, Isono Y, Iwaka H,…, Yoshiki T.
Clin Chem 2004; 50(5):857-866.
How are we going to discover new cancer biomarkers? A proteomic approach for bladder cancer.
Editorial. Eftherios P. Diamandis
Clin Chem 2003; 50(5):794-795.

BACKGROUND: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. METHODS: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range two-dimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. RESULTS: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P = 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80-91%) for bladder cancer in the samples tested. CONCLUSIONS: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the identification of urinary CRT. The diagnostic accuracy of urinary CRT for bladder cancer appears comparable to that of Food and Drug Administration-cleared urinary markers, but further studies are needed to determine its diagnostic role.

A handful of cancer biomarkers are currently used routinely for population screening, disease diagnosis, prognosis, monitoring of therapy, and prediction of therapeutic response. Unfortunately, most of these biomarkers suffer from low sensitivity, specificity, and predictive value, particularly when applied to rare diseases in population screening programs. Thus, for the classic cancer biomarkers much is left to be desired in terms of clinical applicability. We need new cancer biomarkers that will further enhance our ability to diagnose, prognose, and predict therapeutic response in many cancer types. Because biomarkers can be analyzed relatively noninvasively and economically, it is worth investing in discovering more biomarkers in the future. The completion of the Human Genome Project has raised expectations that the knowledge of all genes and proteins will lead to identification of many candidate biomarkers for cancer and other diseases. These predictions still need to be realized. The prevailing view among specialists is that the most powerful single cancer biomarkers may have already been discovered. Likely, in the future we will discover biomarkers that are less sensitive or specific but could be used in panels, in combination with powerful bioinformatic tools, to devise diagnostic algorithms with improved sensitivity and specificity. These efforts are currently in progress1.

  1. Stephan C, Vogel B, Cammann H, Lein M, Klevecka V, Sinha P, et al. [An artificial neural network as a tool in risk evaluation of prostate cancer. Indication for biopsy with the PSA range of 2–20 microg/l]. Urologe A 2003; 42:1221–9.

In this issue of Clinical Chemistry, Kageyama et al. propose proteomic analysis of urine as a new way to identify bladder cancer biomarkers. Previously, Celis et al. 2 used two-dimensional gel electrophoresis and developed a comprehensive database for bladder cancer profiles of both transitional and squamous cell carcinomas. Through their studies, Kageyama et al. were able to identify a potential tumor marker, calreticulin, which is found in the urine of patients with bladder carcinoma. The authors used a differential display method of bladder cancer vs healthy urothelial tissue and mass spectrometry to identify proteins that are increased in cancer tissue. In addition to calreticulin, an endoplasmic reticulum chaperone, they found nine other candidate proteins that could constitute new biomarkers for bladder carcinoma. The authors confirmed their data with quantitative Western blot analysis, immunoprecipitation, and immunohistochemistry. Their reported sensitivity and specificity were 73% and 86%, respectively, similar to the values reported for other biochemical bladder markers. However, the diagnostic accuracy of their test was vulnerable to urinary tract infections3.

3 Positive correlations were found among the appearance of adenylate kinase activity in the urine and the existence of bacteriuria, the Fairley test, and other criteria of urinary infection. Since the adenylate kinase isozymes of human tissues are organ specific and can be distinguished from one another, the appearance of adenylate kinase isozymes in urine was used in this study to identify the existence of infection in bladder or kidney. The findings suggest the usefulness of measuring the appearance of urinary adenylate kinase isozymes for the purpose of detection and differential diagnoses of urinary infections, particularly since adenylate kinase is absent or found in low concentrations in urine and serum under normal conditions.

Currently, potential bladder tumor markers can be used in various clinical scenarios, including4:

  • Serial testing for earlier detection of recurrence;
    • Complementary testing to urine cytology to improve the detection rate;
    • Providing a less expensive and more objective alternative

to the urine cytology test; and
• Directing the cytoscopic evaluation of patient followup.

The gold standard for the detection of urothelial neoplasia is cytologic examination of urothelial cells from voided urine, urinary bladder washings, and urinary tract brushing specimens in combination with cystoscopic examination5,6.

  1. Celis A, Rasmussen HH, Celis P, Basse B, Lauridsen JB, Ratz G, et al. Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities. Electrophoresis 1999;20:355–61.
  2. Bernstein LH, Horenstein JM, and Russell PJ. Urinary adenylate kinase and urinary infections. J Clin Microbiol. 1983 Sep; 18(3): 578–584
  3. Fritsche HA. Bladder cancer and urine tumor marker tests. In: Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK. Tumor markers: physiology,pathobiology, technology and clinical applications. Washington: AACC Press, 2002; 281–6.
  4. Bailey MJ. Urinary markers in bladder cancer. BJU Int 2003; 91:772–3
  5. Eissa S, Kassim S, El-Ahmady O. Detection of bladder tumours: role of cytology, morphology-based assays, biochemical and molecular markers. Curr Opin Obstet Gynecol 2003;15:395–403

Current guidelines suggest that low-risk patients should be surveyed once a year with cystoscopy and high-risk patients at 3-month intervals. Currently, cystoscopy is always combined with VUC. Because, as mentioned earlier, new urinary bladder tests such as BTA or NMP22 could detect lower-grade disease recurrence with higher sensitivity than VUC, it could be worthwhile to consider including one or more of these tests in the routine follow-up of patients with bladder carcinoma. However, large prospective studies will be necessary to test the clinical utility of these assays against cytology.  Multiplexed proteomic analysis of oxidation and concentrations of CSF proteins in Alzheimer’s disease

Korolainen MA, Nyman TA, Nyyssonen P, Hartikainen ES, Pirttila Y.
Clin Chem 2007; 53(4):657-665.

Carbonylation is an irreversible oxidative modification of proteins that has been linked to various conditions of oxidative stress, aging, physiological disorders, and disease. Increased oxidative stress is thus also considered to play a role in the pathogenesis of age-related neurodegenerative disorders such as Alzheimer disease (AD). In addition, it has recently become evident that the response mechanisms to increased oxidative stress may depend on sex. Several oxidized carbonylated proteins have been identified in plasma and brain of AD patients by use of 2-dimensional oxyblotting.

Signals for beta-trace, lambda chain, and transthyretins were decreased in probable AD patients compared with controls. The only identified protein exhibiting an increased degree of carbonylation in AD patients was lambda chain. The concentrations of proteins did not generally differ between men and women; however, vitamin D-binding protein, apolipoprotein A-I, and alpha-1-antitrypsin exhibited higher extents of carbonylation in men.

None of the brain-specific proteins exhibited carbonylation changes in probable AD patients compared with age-matched neurological controls showing no cognitive decline. The carbonylation status of proteins differed between women and men. Two-dimensional multiplexed oxyblotting is applicable to study both the concentrations and carbonylation of cerebrospinal fluid proteins.  The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients

Jin-Moo Lee, Kaj Blennow, Niels Andreasen, Omar Laterza, Vijay Modur, Jitka Olander, Feng Gao, Matt Ohlendorf, and Jack H. Ladenson
Clinical Chemistry  2008; 54:10 1617–1623

BACKGROUND: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid- peptide (A1–42), total Tau (tTau), and hyperphosphorylated Tau (pTau). METHODS: Using ELISA, we measured concentrations of A1–42, tTau, pTau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. RESULTS: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/L] compared with controls [244 (142.5) ng/L]. Although the diagnostic performance of VLP-1 alone was comparable to that of A, tTau, or pTau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE 4/4 genotype [599 (240) ng/L] compared with 3/4 [376 (127) ng/L] and 3/3 [280 (115.5) ng/L] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with pTau (r 0.809) and tTau (r 0.635) but not A1–42 (r 0.233). VLP-1 was the only biomarker that correlated with MMSE score (r 0.384, P 0.030). CONCLUSIONS: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD.

The diagnosis of Alzheimer disease (AD),6 the most common form of dementia in Western countries, is largely based on historical and clinical criteria. Although many studies report a reasonably high degree of diagnostic accuracy (80%–90%), these studies often include patients with advanced disease evaluated at specialized centers (1 ). At present, postmortem examination of brain tissue is the only tool for definitive diagnosis. Therefore, the development of a biomarker for AD would aid greatly in the diagnosis of this disease. In addition, such a marker could potentially be used to measure efficacy in future therapeutic trials. Most studies of AD biomarkers have focused on known pathological substrates for the disease. Amyloid plaques and neurofibrillary tangles are pathological hallmarks of AD (2 ) and primarily comprise abnormally aggregated endogenous proteins. Amyloid plaques (extracellular proteinaceous aggregates) are principally composed of the amyloid- peptide (A), a 38 – to 42–amino acid peptide fragment of the amyloid precursor protein (APP). The major species, the 42– amino acid peptide (A1–42) (3, 4 ), is significantly decreased in the cerebrospinal fluid (CSF) of patients with AD (5– 8 ). Neurofibrillary tangles are intraneuronal protein aggregates found mainly in neurites and primarily composed of hyperphosphorylated Tau (pTau), a microtubule-associated protein.

Fig. 1. CSF VLP-1 values in AD patients and controls. Scatter plot of CSF VLP-1 values in control vs AD patients. The line within the box represents the median value, the box encompasses 25th to 75th percentiles, and the error bars encompass the 10th to 90th percentiles. A significant difference was found in control vs AD patients (P 0.001, Student t-test).

To see if VLP-1 provides utility to the diagnosis of AD beyond the contribution of A, tTau, or pTau alone, we performed a ROC analysisfor each individual biomarker alone compared to the combination of all biomarkers. The AUCs for VLP-1, A, tTau, pTau, and an optimum linear combination of all biomarkers are shown in Fig. 2. AUCs were similar between all biomarkers individually; however, the linear combination of all biomarkers resulted in an approximately 5% improvement (Fig. 2).

To examine possible relationships between CSF VLP-1 values and patient characteristics, we performed correlation analyses between VLP-1 and age, disease duration, MMSE, and the number of APOE 4 alleles. VLP-1 correlated with MMSE and the number of APOE 4 alleles (Fig. 3A). None of the other biomarkers correlated with MMSE in this patient population (A1–42, r 0.350, P 0.497; tTau, r 0.295, P 0.100; pTau, r 0.202, P 0.264). To further examine the relationship between APOE genotype and CSF VLP-1 concentrations, we calculated mean CSF VLP-1 values by different genotypes. APOE 4/4 individuals had the highest concentrations, followed by 3/4 and 3/3 individuals (Fig. 3B).

To examine if VLP-1 concentrations in the CSF were related to values of the other biomarkers studied, we performed correlations between VLP-1 and tTau, pTau, or A1–42 using data from both AD patients and controls (Fig. 4). VLP-1 and pTau showed the greatest correlation (r 0.809) (Fig. 4C), whereas A1–42 did not correlate with VLP-1 (Fig. 4A, r 0.233). Individual correlations for AD patients analyzed separately from controls were also performed, and revealed results similar to that of the total patient population: VLP-1 vs A1–42 was not statistically significant (r 0.29671 and 0.1698 in AD and controls, respectively), whereas VLP-1 vs tTau (r 0.6221 and 0.7247 in AD and controls) and pTau (r 0.8747 and 0.6227 in AD and controls) were significantly correlated in the AD and control populations analyzed separately.

Dementia severity appears to correlate with the number of neurofibrillary tangles, but not to the degree of plaque deposition (13 ). The close correlation between VLP-1 and pTau concentrations in the CSF of AD patients is consistent with these findings, as is the lack of correlation with A. There are several limitations to this study. First, the number of patients in both control and disease groups is limited. Further studies will be needed to confirm our findings in larger, more well-characterized populations. Second, because the diagnosis of AD was made by clinical criteria, there will undoubtedly be a small but significant group of patients that were misdiagnosed (10%–20%) (1 ). This may account for some of the overlap in values for CSF biomarkers. ApoE genotyping in the control group might help with this diagnostic uncertainty. A much more rigorous study would require autopsy confirmation of diagnosis. Third, our study is limited to a comparison of VLP-1 concentrationsin AD patients vs controls, a situation thatis unlikely to occur clinically. A more relevant comparison should be made across patients carrying the differential diagnosis of dementia. Finally, our CSF samples represent a single snapshot in AD pathogenesis; further studies will be required to understand the time course or biomarker evolution with disease pathogenesis. Determination of non-α1-antichymotrypsin-complexed PSA as an indirect measurement of free PSA: analytical performance and diagnostic accuracy.

Wesselin S, Dtephan C, Semjonow A,…, Jung K.
Clin Chem 2003;49(6):887-894.

Background: A new assay measures prostate-specific antigen (PSA) not complexed to α1-antichymotrypsin (nACT-PSA) after removing PSA complexed to ACT by use of anti-ACT antibodies. We evaluated nACT-PSA and its ratio to total PSA (tPSA) as alternatives to free PSA (fPSA) and its ratio to tPSA in differentiating prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in patients with tPSA of 2–20 μg/L. Methods: PSA in serum of 183 untreated patients with PCa and 132 patients with BPH was measured retrospectively on the chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays. The nACT-PSA fraction was determined with a prototype assay measuring the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent.
Results: nACT-PSA was higher than fPSA in samples with fPSA concentrations <1 μg/L but lower in samples with >1 μg/L fPSA. The median ratios of fPSA/tPSA and of nACT-PSA/tPSA were significantly different between patients with BPH and PCa (19.4% vs 12.2% and 17.4% vs 13.0%, respectively). Within the tPSA ranges tested (2–20, 2–10, and 4–10 μg/L), areas under the ROC curves for the fPSA/tPSA ratios were significantly larger than those for nACT-PSA/tPSA. In the tPSA ranges <10 μg/L, the areas under the ROC curves for fPSA/tPSA were significantly larger than those for tPSA, whereas the areas for nACT-PSA/tPSA were not. At decision limits for 95% sensitivity and specificity, both ratios significantly increased specificity and sensitivity, respectively, compared with tPSA, but the fPSA/tPSA ratio showed higher values. Conclusions: nACT-PSA and its ratio to tPSA provide lower diagnostic sensitivity and specificity than fPSA/tPSA. The fPSA/tPSA ratio represents the state-of-the-art method for differentiating between PCa and BPH. Ultrasensitive densitometry detection of cytokines with nanoparticle-modified aptamers

Li yuan-Yuan, Zhang C, Li Bo-Sheng, …, Xu Shun-Quing
Clin Chem 2007; 53(6):1061-1066

Background: Aptamers mimic properties of antibodies and sometimes turn out to be even better than antibodies as reagents for assays. We describe the establishment of an ultrasensitive densitometry method for cytokine detection by nanoparticle (NP)-modified aptamers. Methods: The assay simultaneously uses a gold NP–modified aptamer and a biotin-modified aptamer to bind to the target protein, forming a sandwich complex. The absorbance signal generated by the aptamer-protein complex is amplified and detected with a microplate reader. Results: The assay for platelet-derived growth factor B-chain homodimer (PDGF-BB) was linear from 1 fmol/L to 100 pmol/L (R2 = 0.9869). The analytical detection limit was 83 amol/L. The intraassay and interassay imprecision (CVs) was ≤7.5%. Serum concentrations of PDGF-BB determined with the gold NP–modified aptamer assay and with ELISA were not significantly different. Conclusions: The gold NP–modified aptamer assay provides a fast, convenient method for cytokine detection and improves the detection range and the detection limit compared with ELISA.  Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker.

Melle C, Ernst G, Escher N, Hartmann D,…, von Eggeling F.
Clin Chem 2007; 53(4):629-635.

Background: Patients with pancreatic adenocarcinomas have a poor prognosis because of late clinical manifestation and the tumor’s aggressive nature. We used proteomic techniques to search for markers of pancreatic carcinoma. Methods: We performed protein profiling of microdissected cryostat sections of 9 pancreatic adenocarcinomas and 10 healthy pancreatic tissue samples using ProteinChip technology (surface-enhanced laser desorption/ionization). We identified proteins by use of 2-dimensional gel electrophoresis, peptide fingerprint mapping, and immunodepletion and used immunohistochemistry for in situ localization of the proteins found. We used ELISA to quantify these proteins in preoperative serum samples from 35 patients with pancreatic cancer and 37 healthy individuals. Results: From among the differentially expressed signals that were detected by ProteinChip technology, we identified 2 proteins, DJ-1 and heat shock protein 27 (HSP27). We then detected HSP27 in sera of patients by use of ELISA, indicating a sensitivity of 100% and a specificity of 84% for the recognition of pancreatic cancer. Conclusions: The detection of DJ-1 and HSP27 in pure defined tissue and the retrieval of HSP27 in serum by antibody-based methods identifies a potential marker for pancreatic cancer.

1.1.7  Mass Spectrometry Methods LC-MS/MS quantification of Zn-α2 glycoprotein: A potential serum biomarker for prostate cancer

Bondar OP, Barnidge DR, KKlee EW, Davis BJ, Klee GG
Clin Chem 2007; 53(4):673-678 http://dx.doi.org:/10.1373/clinchem.2006.079681

LC-MS/MS – tandem mass spectrometry

Background: Zn-α2 glycoprotein (ZAG) is a relatively abundant glycoprotein that has potential as a biomarker for prostate cancer. We present a high-flow liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for measuring serum ZAG concentrations by proteolytic cleavage of the protein and quantification of a unique peptide. Methods: We selected the ZAG tryptic peptide 147EIPAWVPEDPAAQITK162 as the intact protein for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Standards using recombinant ZAG in bovine serum albumin, 50 g/L, and a pilot series of patient sera were denatured, reduced, alkylated, and digested with trypsin. The concentration of ZAG was calculated from a dose–response curve of the ratio of the relative abundance of the ZAG tryptic peptide to internal standard. Results: The limit of detection for ZAG in serum was 0.08 mg/L, and the limit of quantification was 0.32 mg/L with a linear dynamic range of 0.32 to 10.2 mg/L. Replicate digests from pooled sera run during a period of 3 consecutive days showed intraassay imprecision (CV) of 5.0% to 6.3% and interassay imprecision of 4.4% to 5.9%. Mean (SD) ZAG was higher in 25 men with prostate cancer [7.59 (2.45) mg/L] than in 20 men with nonmalignant prostate disease [6.21 (1.65) mg/L, P = 0.037] and 6 healthy men [3.65 (0.71) mg/L, P = 0.0007]. Conclusions: The LC-MS/MS assay can be used to evaluate the clinical utility of ZAG as a cancer biomarker. A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples.

Lopez MF, Mikulskis A, Kuzdzal S, Golenko E,…, Fishman D.
Clin Chem 2007; 53(6):1067-1074.


Background: Most cases of ovarian cancer are detected at later stages when the 5-year survival is ∼15%, but 5-year survival approaches 90% when the cancer is detected early (stage I). To use mass spectrometry (MS) of serum proteins for early detection, a seamless workflow is needed that provides an opportunity for rapid profiling along with direct identification of the underpinning ions. Methods: We used carrier protein–bound affinity enrichment of serum samples directly coupled with MALDI orthagonal TOF MS profiling to rapidly search for potential ion signatures that contained discriminatory power. These ions were subsequently directly subjected to tandem MS for sequence identification. Results: We discovered several biomarker panels that enabled differentiation of stage I ovarian cancer from unaffected (age-matched) patients with no evidence of ovarian cancer, with positive results in >93% of samples from patients with disease-negative results and in 97% of disease-free controls. The carrier protein–based approach identified additional protein fragments, many from low-abundance proteins or proteins not previously seen in serum. Conclusions: This workflow system using a highly reproducible, high-resolution MALDI-TOF platform enables rapid enrichment and profiling of large numbers of clinical samples for discovery of ion signatures and integration of direct sequencing and identification of the ions without need for additional offline, time-consuming purification strategies.  Mass Spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.

Kemna EHJM, Tjalsma H, Podust VN, Swinkels DW.
Clin Chem 2007; 53(4):620-628.


Background: Discovery of the central role of hepcidin in body iron regulation has shed new light on the pathophysiology of iron disorders. Information is lacking on newer analytical approaches to measure hepcidin in serum and urine. Recent reports on the measurement of urine and serum hepcidin by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) necessitate analytical and clinical evaluation of MS-based methodologies. Methods: We used SELDI-TOF MS, immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, we investigated analytical reproducibility and biological and preanalytical variation for both serum and urine on Normal Phase 20 and Immobilized Metal Affinity Capture 30 ProteinChip arrays. We obtained samples from healthy controls and patients with documented iron-deficiency anemia, inflammation-induced anemia, thalassemia major, and hereditary hemochromatosis. Results: Proteomic techniques showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. The interarray CV was 15% to 27%, and interspot CV was 11% to 13%. Preliminary studies showed that hepcidin-25 differentiated disorders of iron metabolism. Urine hepcidin is more affected by multiple freeze-thaw cycles and storage conditions, but less influenced by diurnal variation, than is serum hepcidin. Conclusion: SELDI-TOF MS can be used to measure hepcidin in both serum and urine, but serum requires a standardized sampling protocol.  Current state and future directions of neurochemical biomarkers for Alzheimer’s disease.

In this comprehensive review, we summarize the current state-of-the-art of neurochemical biomarkers for Alzheimer’s disease. Predominantly, these biomarkers comprise cerebrospinal fluid biomarkers directly related to the pathophysiology of this disorder (such as amyloid beta protein, tau protein). We particularly pay attention to the innovations in this area that have been made in technological aspects during the past 5 years (e.g., multiplex analysis of biomarkers, proteomics), to the discovery of novel, potential biomarkers (e.g., amyloid beta oligomers, isoprostanes), and to the extension of this research towards identification of biomarkers in plasma.  Use of SELDI-TOF mass spectrometry for identification of new biomarkers: potential and limitations.

Surface-enhanced laser desorption time of flight mass spectrometry (SELDI-TOF-MS) is an important proteomic technology that is immediately available for the high throughput analysis of complex protein samples. Over the last few years, several studies have demonstrated that comparative protein profiling using SELDI-TOF-MS breaks new ground in diagnostic protein analysis particularly with regard to the identification of novel biomarkers. Importantly, researchers have acquired a better understanding also of the limitations of this technology and various pitfalls in biomarker discovery. Bearing these in mind, great emphasis must be placed on the development of rigorous standards and quality control procedures for the pre-analytical as well as the analytical phase and subsequent bioinformatics applied to analysis of the data. To avoid the risk of false-significant results studies must be designed carefully and control groups accurately selected. In addition, appropriate tools, already established for analysis of highly complex microarray data, need to be applied to protein profiling data. To validate the significance of any candidate biomarker derived from pilot studies in appropriately designed prospective multi-center studies is mandatory; reproducibility of the clinical results must be shown over time and in different diagnostic settings. SELDI-TOF-MS-based studies that are in compliance with these requirements are now required; only a few have been published so far. In the meantime, further evaluation and optimization of both technique and marker validation strategies are called for before MS-based proteomic algorithms can be translated into routine laboratory testing.

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Preface to Metabolomics as a Discipline in Medicine

Author: Larry H. Bernstein, MD, FCAP


The family of ‘omics fields has rapidly outpaced its siblings over the decade since
the completion of the Human Genome Project.  It has derived much benefit from
the development of Proteomics, which has recently completed a first draft of the
human proteome.  Since genomics, transcriptomics, and proteomics, have matured
considerably, it has become apparent that the search for a driver or drivers of cellular signaling and metabolic pathways could not depend on a full clarity of the genome. There have been unresolved issues, that are not solely comprehended from assumptions about mutations.

The most common diseases affecting mankind are derangements in metabolic
pathways, develop at specific ages periods, and often in adulthood or in the
geriatric period, and are at the intersection of signaling pathways.  Moreover,
the organs involved and systemic features are heavily influenced by physical
activity, and by the air we breathe and the water we drink.

The emergence of the new science is also driven by a large body of work
on protein structure, mechanisms of enzyme action, the modulation of gene
expression, the pH dependent effects on protein binding and conformation.
Beyond what has just been said, a significant portion of DNA has been
designated as “dark matter”. It turns out to have enormous importance in
gene regulation, even though it is not transcriptional, effected in a
modulatory way by “noncoding RNAs.  Metabolomics is the comprehensive
analysis of small molecule metabolites. These might be substrates of
sequenced enzyme reactions, or they might be “inhibiting” RNAs just
mentioned.  In either case, they occur in the substructures of the cell
called organelles, the cytoplasm, and in the cytoskeleton.

The reactions are orchestrated, and they can be modified with respect to
the flow of metabolites based on pH, temperature, membrane structural
modifications, and modulators.  Since most metabolites are generated by
enzymatic proteins that result from gene expression, and metabolites give
organisms their biochemical characteristics, the metabolome links
genotype with phenotype.

Metabolomics is still developing, and the continued development has
relied on two major events. The first is chromatographic separation and
mass  spectroscopy (MS), MS/MS, as well as advances in fluorescence
ultrasensitive optical photonic methods, and the second, as crucial,
is the developments in computational biology. The continuation of
this trend brings expectations of an impact on pharmaceutical and
on neutraceutical developments, which will have an impact on medical
practice. What has lagged behind, and may continue to contribute to the
lag is the failure to develop a suitable electronic medical record to
assist the physician in decisions confronted with so much as yet,
hidden data, the ready availability of which could guide more effective
diagnosis and management of the patient. Put all of this together, and
we can meet series challenges as the research community
interprets and integrates the complex data they are acquiring.


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