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July 2011

Editor:
Michael Bissell, MD, PhD, MPH

Evaluation of new peptide assay technology Evaluation of new peptide assay technology

The quantitative measurement of biomarkers is central to the pharmacodynamic assessment of drug candidates during pharmaceutical development and to clinical disease management. Protein biomarkers constitute a major focus within this field, and analytical modalities for their measurement are evolving and maturing. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been explored in recent years for protein biomarker quantification. Most published approaches measure one or several peptides, which are enzymatically released from the target protein of interest as part of the assay procedure. Quantification is typically based on molar equivalency between the surrogate peptide and protein. Hybrid immunoaffinity-mass spectrometric methods have been developed to address the detection-limit challenges associated with measuring low-abundance protein biomarkers in the low- and sub-microgram per liter range. Antibody capture can occur at the protein or peptide level. The most appropriate approach for achieving the analytical aim depends on a multitude of factors, including reagent availability, sample preparation platforms, throughput requirements, and the followup method for biomarker quantification. A promising immunoaffinity strategy for protein biomarker quantification is stable isotope standards and capture by antipeptide antibodies. This strategy includes enriching an enzymatically derived peptide and its synthetic stable isotope-labeled analog using an antipeptide antibody followed by LC-MS/MS detection, executed primarily with selected-reaction–monitoring mass spectrometry. Using a different combination of chromatography and mass spectrometry modalities, other groups have focused on hyphenating relatively large-capacity, high-flow peptide immunoaffinity chromatography online with conventional LC-MS/MS. Assays based on this technology have been validated for clinical use and show the robustness necessary for routine implementation in clinical protein biomarker investigations. Depending on the antibody and relevant assay parameters, the peptide immunoaffinity columns can be used for hundreds of runs to a few thousand runs, contributing to overall robustness. The authors reported on an online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. They validated this approach by developing an assay for salivary pepsin/pepsinogen, which has been reported to be detectable in the saliva of some patients with gastroesophageal reflux disease and which correlates with proximal reflux episodes, detected using 24-hour pHmetry. For their study, saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence DRANNQVGLAPVA was linked to nanoflow liquid chromatography and selected-reaction–monitoring mass spectrometry. The authors used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers. The authors found that heat inactivation at 100°C for 25 minutes stabilized the target peptide. The final assay had less than 15 percent interassay relative error and less than 15 percent interassay coefficient of variation across a range of 4.08 to 2,980 pmol/L human pepsinogen (0.165–120 µg/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers, ranging from 4.3 to 16.6 pmol/L (0.17–0.67 µg/L) total salivary pepsin/pepsinogen. The authors concluded that this assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It has the potential to deliver additional data on the occurrence of pepsin/pepsinogen in saliva with greater confidence than that provided by previous methodologies.

Neubert H, Gale J, Muirhead D. Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen. Clin Chem. 2010;56:1413–1423.

Correspondence: Hendrik Neubert at hendrik.neubert@pfizer.com

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Novel Treponema pallidum serologic tests Novel Treponema pallidum serologic tests

Syphilis, once known as “the Great Pox,” continues to challenge clinicians with its nuances in diagnosis and management. On the basis of the Wasserman test, introduced more than 100 years ago, syphilis diagnosis continues to rely on serologic assays because Treponema pallidum cannot be cultured in vitro. Furthermore, direct visualization of the spirochete requires lesions and fluorescent antibodies or a dark-field microscope, neither of which may be readily available. T. pallidum nucleic acid amplification tests are not widely available to clinical laboratories. Therefore, serologic tests are the foundation of syphilis management, and knowledge of their diagnostic limitations is critical for clinicians. The FDA has cleared several syphilis serologic tests as diagnostic, confirmatory, and blood donor screening tests. However, a greater number of syphilis tests are now commercially available, primarily because of less stringent requirements for their development in other countries. In contrast to older methods, such as the rapid plasma reagin (RPR) test, which use phospholipid (nontreponemal) antigens, newer serologic tests use specific T. pallidum antigens. These new technologies have flooded international markets because of their level of automation. Although most have not been cleared by the FDA, these assays may be used after validating their performance in comparison with reference standards. The new treponemal-specific assays have displaced nontreponemal tests for screening in some laboratories and have the potential to confuse clinical management. The authors reviewed new serologic tests that have been widely evaluated for screening noncongenital syphilis. Many new diagnostic methods for syphilis screening have been developed using specific treponemal antigens and novel formats, including rapid point-of-care tests, enzyme immunoassays, and chemiluminescence assays. Both sensitive and specific, new screening tests detect anti-treponemal IgM and IgG antibodies by use of wild-type or recombinant T. pallidum antigens. However, these tests cannot distinguish between recent and remote or treated versus untreated infections. Furthermore, the screening tests must be confirmed with nontreponemal tests. The use of treponemal tests for screening and nontreponemal serologic tests as confirmatory tests is a reversal of long-held practice. Clinicians need to understand the science behind these tests to use them properly in syphilis management.

Sena AC, White BL, Sparling PF. Novel Treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century. Clin Infect Dis. 2010;51:700–708.

Correspondence: Arlene C. Sena at idrod@med.unc.edu

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Obesity and prostate cancer risk in African-Americans Obesity and prostate cancer risk in African-Americans

Prostate cancer continues to be a major public health concern—an estimated 186,320 new cases of prostate cancer were diagnosed in 2008. Despite the significant role of obesity and prostate cancer in public health, the relationship between these disease states remains unclear. A recent study from Columbia University evaluated 964 patients after radical prostatectomy, focusing on their body mass index. The study found that despite higher risk disease, obese patients did not have higher prostate-specific antigen (PSA) recurrence rates. This study contradicted one that analyzed 1,106 patients after radical prostatectomy and found that obesity resulted in higher grade tumors and higher biochemical failure rates. Based on the lack of consensus about the effect of obesity on prostate cancer, continued study is warranted. African-American race has been associated with increased prostate cancer incidence and mortality rates. Many factors, including socio-economic status, access to health care, and obesity, have been linked to a worse prostate cancer prognosis in African-American men. One study reported that African-American men are less likely to maintain regular PSA screening practices, perhaps leading to a delay in diagnosis. Others have concluded that socio-economic factors alone explain the disparity in outcome between African-American and Caucasian men. Furthermore, obesity in African-American men has been associated with a particularly poor prognosis. The authors conducted a study in which they analyzed the link between African-American race and obesity in patients with impalpable prostate cancer and a diagnostic PSA level of less than 10 ng/mL. The primary goal of the study was to assess racial disparities in PSA recurrence rates among obese patients. To analyze the relationship between African-American race and obesity in men with prostate cancer, the authors identified from the Duke Prostate Center database 4,196 patients who underwent radical prostatectomy from 1988 to 2008. They stratified by race and then analyzed a subset of 389 patients (20.9 percent African-American and 79.1 percent non-African-American) with a body mass index of 30 kg/m2 or more, T1c disease, and a PSA level of less than 10 ng/mL. Age at surgery, race, surgical margin status, pathological tumor stage (pT2, pT3/4), pathological Gleason sum (less than 7, 3+4, 4+3, more than 7), extracapsular extension, seminal vesicle invasion, and tumor percentage were assessed by univariate analysis followed by Cox regression analysis. The authors found that in the entire cohort, 143 (38.1 percent) African-American men were obese, compared with 509 (25 percent) of the non-African-American men. And African-American men, when compared to non-African-American men, had a significantly higher tumor percentage (15 versus 10 percent; P=0.002), as well as a greater proportion of pT3/4 disease (45.1 versus 26.2 percent; P=0.039), pathological Gleason sum of seven or higher (70.7 versus 50.5 percent; P=0.003), positive extracapsular extension (37.8 versus 23.1 percent; P=0.007), and positive surgical margin (52.4 versus 36.8 percent; P=0.010). African-American men had a greater risk of PSA recurrence on Kaplan Meier (P=0.004) and Cox regression analysis (P=0.040; hazard ratio, 1.72). The authors concluded that a greater proportion of African-American men were obese in this cohort. Obese African-American men with impalpable cancer and a PSA level of less than 10 ng/mL have a higher risk of pathological features than obese non-African-American men, as well as a higher risk of PSA recurrence. Obesity might be responsible for the racial disparity seen in prostate cancer.

Caire AA, Sun L, Polascik TJ, et al. Obese African-Americans with prostate cancer (T1c and a prostate-specific antigen, PSA, level of <10 ng/mL) have higher-risk pathological features and a greater risk of PSA recurrence than non-African-Americans. Br J Urol Int. 2010;106:1157–1160.

Correspondence: Judd W. Moul at judd.moul@duke.edu

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Expression of p53, CEA, and CA 19.9 in cancer of the gallbladder Expression of p53, CEA, and CA 19.9 in cancer of the gallbladder

Gallbladder cancer is the most common gastrointestinal cancer in women of north India. Although precursor epithelial lesions in gallbladder cancer (GBC) are known, the role of xanthogranulomatous (XG) inflammation in the pathogenesis of GBC is not known. To analyze the role of precursor lesions in the pathogenesis of this cancer, the authors studied the immunohistochemical expression of p53, carcino-embryonic antigen (CEA), and carbohydrate antigen 19.9 (CA 19.9) in GBC, chronic cholecystitis, XG cholecystitis, and precursor lesions. The authors included in their study 51 patients with GBC, 68 with chronic cholecystitis, 42 with XG cholecystitis, and 10 with normal gallbladders. All cases were evaluated for the presence of precursor lesions, and immunohistochemistry was performed. The authors found p53 immunoreactivity in 55 percent of GBC, 32 percent of dysplasia with malignancy, and 14 percent of dysplasia with chronic cholecystitis. Sixteen percent of those with GBC had associated XG inflammation. Normal and metaplastic epithelium in chronic cholecystitis and XG cholecystitis did not express p53. CEA expression was apical in normal and inflammatory gallbladders, while cytoplasmic focal to diffuse positivity was seen in 82 percent of GBC. CA 19.9 expression was seen in all cases of normal and inflammatory gallbladders. However, foci of antral metaplasia were negative. Seventy-five percent of GBC expressed CA 19.9; all negative cases were high grade on histology. The authors concluded that altered CEA expression is seen in GBC as compared to normal and inflammatory gallbladders. Loss of expression of CA 19.9 in antral metaplasia and poorly differentiated GBC may indicate that it is a marker of biliary differentiation. Overexpression of p53 in GBC and dysplasia associated with malignancy and chronic cholecystitis suggests that p53 mutation and dysplasia are early events in the evolution of GBC. Epithelial metaplasia and XG inflammation are often associated with GBC but do not appear to play a role in its pathogenesis through the p53 pathway.

Agrawal V, Goel A, Krishnani N, et al. p53, carcinoembryonic antigen and carbohydrate antigen 19.9 expression in gall bladder cancer, precursor epithelial lesions and xanthogranulomatous cholecystitis. J Postgrad Med. 2010;56:262–266

Correspondence: Dr. Vinita Agrawal at vinita@sgpgi.ac.in

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Opiate-positive drug screen in a pediatric patient Opiate-positive drug screen in a pediatric patient

A three-year-old girl was transferred from an outside hospital to the emergency department after being found shivering and unattended outside a public shopping area in winter. She presented with decreased mental status and possible hypothermia; she was lethargic and slipped in and out of consciousness. No cause for this alteration in her mental state was obvious. A physical examination revealed multiple abrasions and bruises. The patient had a temperature of 37°C, blood pressure of 134/74 mmHg, heart rate of 103 beats per minute, and respiratory rate of 24 breaths per minute. Computed tomography scans, a skeletal survey, and ophthalmologic examination did not reveal additional injuries. The patient received an intravenous bolus of the opioid antagonist naloxone (Narcan) shortly after arriving in the emergency department. Routine hematology and chemistry tests revealed decreased serum urea nitrogen and increased lactate, aspartate aminotransferase, and alanine aminotransferase. Serum acetaminophen and salicylate were not detected, and blood carboxyhemoglobin was not increased. Urine and stool cultures were negative, as was a nasal swab for methicillin-resistant Staphylococcus aureus. Toxicologic screening of a urine sample collected when the girl arrived at the emergency department detected no amphetamines, barbiturates, benzodiazepines, cocaine metabolite, opiates (codeine and morphine), tetrahydrocannabinol (marijuana), or methodone. A gas chromatographic screen for volatile substances in the patient’s serum did not detect ethanol, methanol, or isopropyl alcohol but was positive for acetone. The patient was admitted to a pediatric floor for further monitoring. Early the next day, clinicians requested a second urine toxicology immunoassay screen. This sample was positive for opiates at a cutoff of 300 µg/L. Confirmatory testing by gas chromatography-mass spectrometry was negative for codeine, hydrocodone, oxycodone, morphine, hydromorphone, and oxymorphone (100 µg/L cutoff). A repeat immunoassay in the authors’ laboratory substantiated the original positive opiate result, and further investigation was initiated. Immunoassays used to screen for the presence of drugs of abuse may not be specific for the intended drug or class and may cross-react with other prescription and nonprescription drugs. Confirmatory testing using a second method with greater specificity and lower detection limits is useful when a screening result is questioned or needs to be verified. Confirmatory testing is commonly required in sensitive cases. Both screening and confirmatory tests use cutoff concentrations to distinguish between positive and negative results. The results of drug or drug metabolite tests may be truly negative, or the drugs or their metabolites may be present at concentrations below the cutoff value and, consequently, be reported as negative or not detected. Drugs and drug metabolites that share structural similarities with the target compound may cross-react with the antibodies used in screening immunoassays. Cross-reactivity experiments may be useful in confirming conflicting clinical results. When interpreting toxicology results, clinicians and laboratory scientists should remember that pediatric patients have a different pharmacokinetic profile for many substances than adults.

Straseski JA, Stolbach A, Clarke W. Clinical case study: opiate-positive immunoassay screen in a pediatric patient. Clin Chem. 2010;56:1220–1225.

Correspondence: William Clarke at wclarke@jhmi.edu

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Tandem mass spectrometry screen for VLCAD deficiency Tandem mass spectrometry screen for VLCAD deficiency

A multiplexed analytical approach is possible with the availability of tandem mass spectrometry, which allows users to simultaneously assess fatty acids by their acylcarnitine counterparts. Therefore, disease-specific metabolite profiles can be a means of identifying patients with long- and medium-chain acyl-CoA dehydrogenase deficiencies during newborn screening in the presymptomatic state. However, at least for long-chain defects, it has been recommended that newborn screening be performed within a small time window, between 36 and 72 hours of life, to allow detection of the enzyme defect. This is recommended largely because patients with long-chain acylcarnitine defects may present with a normal acylcarnitine pattern during anabolic conditions when fatty acid oxidation is not induced. In mild phenotypes, this is frequently observed after day three of life. Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (ACADVL, OMIM 609575) is the most common long-chain b-oxidation defect. Since implementing newborn screening and further identifying patients with mild or silent disease, prevalence has been estimated to be one in 50,000 to one in 100,000. This is higher than estimated before the screening era. Most children identified with a confirmed VLCAD deficiency present with milder or asymptomatic phenotypes, without elevated acylcarnitine concentrations in blood, and with normal results on organic acid analysis in urine during anabolism. However, asymptomatic clinical presentations that occur later in life may be the effect of early preventive measures that prevent symptomatic disease, such as avoidance of fasting, emergency protocols during acute illnesses, and a fat-modified and fat-restricted diet. Some individuals may present with a mildly elevated C14:1-carnitine concentration just higher than the cutoff threshold without other long-chain acylcarnitines affected during the initial newborn screening at days two to three of life. Stimulation of fatty acid oxidation during catabolism results in elevated concentrations of long-chain acylcarnitines, and this typically happens during the first days after birth. With increasing amounts of food ingested and the switch to anabolism, the results of a second screening on day five or later may already be normal. Because of the potential of false-positive results in samples in the first screening and the danger of classifying a patient with VLCAD deficiency as normal during a repeated screening test, the authors prospectively investigated 90,338 newborns who had an increased concentration of one or all C14-carnitine derivatives using enzyme and molecular genetic analyses. They performed palmitoyl-CoA oxidation in lymphocytes to define very long-chain acyl-CoA dehydrogenase function. Molecular analysis was then performed in children with residual activities of less than 50 percent. The acylcarnitine pattern on days two to three of life was evaluated thoroughly to define possible discrimination markers. The authors identified 40 newborns with increased C14:1-carnitine (1:2,500). In two newborns, VLCAD deficiency was confirmed with enzyme and molecular analyses (prevalence, 1:50,000). One of the newborns had normal results on a second screening. The combination of absolute acylcarnitine values and acylcarnitine ratios did not allow the newborn to be identified correctly as a patient with VLCAD deficiency. The authors concluded that reliable diagnosis is not feasible with acylcarnitine analysis alone. Enzyme analysis in lymphocytes is a reliable and rapid method for assessing all newborns with VLCAD deficiency and should be performed for all newborns identified during the first screening, regardless of the results of a later acylcarnitine profile.

Spiekerkoetter U, Haussmann U, Mueller M, et al. Tandem mass spectrometry screening for very long-chain acyl-CoA dehydrogenase deficiency: the value of second-tier enzyme testing. J Pediatr. 2010;157:668–673.

Correspondence information not available.

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Comparing provocative tests for pediatric adrenal insufficiency Comparing provocative tests for pediatric adrenal insufficiency

Adrenal insufficiency is a life-threatening condition that may result from primary adrenal failure or from impairment of the hypothala-mic-pituitary adrenal (HPA) axis. It is frequently observed in patients with genetic hypopituitarism and can be predicted on the basis of genetic abnormalities. Similarly, patients with growth hormone (GH) deficiency and structural hypothalamic-pituitary abnormalities are at risk of developing adrenocorticotropic hormone deficiency, as well as a worsening of pituitary-adrenal function caused by progressive deterioration of anterior pituitary function. Although the diagnosis of overt adrenal failure is generally straightforward, identifying asymptomatic patients with subtle dysfunctions of the HPA axis is still a diagnostic challenge. The reference tests for establishing the integrity of the HPA axis rely on response to a strong stimulus, such as the insulin-induced hypoglycemia/insulin tolerance test (ITT), or interruption of negative feedback (overnight metyrapone test). However, these reference tests also have significant drawbacks. The ITT is contraindicated in infants and children with a history of seizures or cardiovascular disease and requires continuous monitoring for possible side effects. The overnight metyrapone test carries a risk of adrenal crisis, and drugs affecting metyrapone clearance may provide unreliable results. Therefore, a need exists for alternative clinical tests that are quicker and safer. A recent meta-analysis showed that the low-dose corticotrophin test was superior to the standard-dose test for diagnosing chronic central adrenal insufficiency in adults and children. However, the low-dose test has not been validated in young children. The authors conducted a study to evaluate the diagnostic ability of the glucagon test to assess adrenal function in children younger than six years of age who had well-defined endocrine and magnetic resonance imaging features and predisposing factors for central adrenal insufficiency. The ITT was regarded as the gold standard test for diagnosing central adrenal insufficiency. The prospective study was conducted in two pediatric endocrinology centers. Enrolled were 48 children (median age, 4.2 years) with GH deficiency confirmed by a peak GH to ITT and arginine of less than 10 µg/L. Twenty-four of the children had normal hypothalamic-pituitary anatomy, while seven had isolated anterior pituitary hypoplasia and 17 structural hypothalamic-pituitary abnormalities at magnetic resonance imaging. Twelve subjects had central adrenal insufficiency defined by a peak cortisol response to ITT of less than 20 µg/dL. All children underwent a glucagon stimulation test with blood sampling for cortisol and glucose (time, zero to 180 minutes) after the administration of 30 µg/kg glucagon. The authors found that the mean peak cortisol after glucagon was not significantly different from that obtained after ITT in the entire cohort (25.9 versus 26 µg/dL; P=0.908), and it was significantly reduced in patients with structural hypothalamic-pituitary abnormalities (P<0.001). Receiver operating characteristic curve analysis showed that the best diagnostic accuracy was obtained with a peak cortisol cutoff to glucagon of 14.6 µg/dL (sensitivity, 66.67 percent; specificity, 100 percent; area under the curve, 0.91; 95 percent confidence interval, 0.82–0.99). Using this cutoff, 91.67 percent of the patients were classified correctly. The authors concluded that this study shows that glucagon is an accurate and safe diagnostic test for adrenal function in young children who are at risk for adrenal insufficiency.

Di Iorgi N, Napoli F, Allegri A, et al. The accuracy of the glucagon test compared to the insulin tolerance test in the diagnosis of adrenal insufficiency in young children with growth hormone deficiency. J Clin Endocrinol Metab. 2010;95:2132–2139.

Correspondence: Dr. Mohamad Maghnie at mohamad.maghnie@oespedale-gaslini.ge.it or mohamad.maghnie@unige.it

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.