Per E. Jørgensen

Reproducibility of mass spectrometry based protein profiles for diagnosis of breast cancer across clinical studies: a systematic review.

Serum protein profiling by mass spectrometry has achieved attention as a promising technology in oncoproteomics. We performed a systematic review of published reports on protein profiling as a diagnostic tool for breast cancer. The MEDLINE, EMBASE, and COCHRANE databases were searched for original studies reporting discriminatory protein peaks for breast cancer as either protein identity or as m/ z values in the period from January 1995 to October 2006. To address the important aspect of reproducibility of mass spectrometry data across different clinical studies, we compared the published lists of potential discriminatory peaks with those peaks detected in an original MALDI MS protein profiling study performed by our own research group. A total of 20 protein/peptide profiling studies were eligible for inclusion in the systematic review. Only 3 reports included information on protein identity. Although the studies revealed a considerable heterogeneity in relation to experimental design, biological variation, preanalytical conditions, methods of computational data analysis, and analytical reproducibility of profiles, we found that 45% of peaks previously reported to correlate with breast cancer were also detected in our experimental study. Furthermore, 25% of these redetected peaks also showed a significant difference between cases and controls in our study. Thus, despite known problems related to reproducibility, we were able to demonstrate overlap in peaks between clinical studies indicating some convergence toward a set of common discriminating, reproducible peaks for breast cancer. These peaks should be further characterized for identification of the protein identity and validated as biomarkers for breast cancer.

Human epidermal growth factor-on molecular forms present in urine and blood.

A sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of human epidermal growth factor (EGF) was employed to study EGF in urine and blood. The EGF/creatinine ratio in urine was significantly higher for women (range and (median); 0.20-0.83 (0.50) nmol EGF/mmol creatinine) than for men (0.17-0.63 (0.30) nmol EGF/mmol creatinine). We were not able to demonstrate EGF in plasma (median plasma EGF < 0.01 nmol/l) whereas serum contained a range and (median) of 0.02-0.31 (0.12) nmol EGF/l. The amount of EGF in serum showed a weak correlation to the platelet count (r = 0.327). EGF was partly purified by affinity chromatography from urine (urine EGF) and from activated platelets in platelet rich plasma (blood EGF). Both blood and urine contained a high molecular weight form of EGF (HMW EGF) as well as 6 kDa EGF. HMW EGF from blood was similar to HMW EGF from urine concerning behaviour upon gel filtration, pI and apparent affinity constant for binding to the EGF receptor. However, HMW EGF constituted approx. 40% of blood EGF but only 10% of urinary EGF. The 6 kDa EGF from both blood and urine contained two isopeptides with pI around 4.40 and 4.15 but in various proportions. The apparent affinity constant for binding to the EGF receptor for blood 6 kDa EGF was 1.8 x 10(10) l/mol compared to 1.0 x 10(10) l/mol for urinary 6 kDa EGF and 0.8 x 10(10) l/mol for HMW EGF from both blood and urine. The present study suggests that the processing of the EGF precursor differs in the blood and in the kidneys and that 6 kDa EGF from blood and urine binds to the EGF receptor with a higher apparent affinity constant than does HMW EGF.

Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer: a case-control study.

Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a reference protein profile. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry.

Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum requires optimized, reproducible methods for handling and deposition of protein samples. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of sample deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties; reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties; reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each sample preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum sample.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum sample. These protein peaks can be part of a possible diagnostic profile, suggesting that this sample preparation method and data acquisition approach is suitable for large-scale analysis of serum samples for protein profiling.

Urinary excretion of epidermal growth factor and Tamm-Horsfall protein in three rat models with increased renal excretion of urine

Epidermal growth factor (EGF) and Tamm-Horsfall protein (THP) are synthesized in the kidneys by the distal tubular cells and excreted into urine. The urinary excretion of these peptides has been suggested as a potential index for distal tubular function. The urinary excretion rates of EGF and THP were examined in three groups of rats with increased renal excretion of urine: uninephrectomy, non-osmotic polyuria and diabetic osmotic polyuria. Twenty-four hour urine samples were obtained after 7, 14 and 21 days. The urinary volume per kidney was doubled in uninephrectomy when compared to controls. There was a seven-fold increase in urinary volume in rats with non-osmotic polyuria and diabetic osmotic polyuria, as compared to controls. Uninephrectomy, non-osmotic polyuria and diabetes all affected the urinary excretion of EGF and THP differently. The EGF excretion in uninephrectomized rats was 60-80% of that of the controls, whereas THP excretion was unchanged, indicating that EGF excretion varied with renal tissue mass. Non-osmotic polyuria caused a five-fold increase in THP excretion but no change in EGF excretion. THP excretion in the diabetic rats was increased three-fold after 21 days when compared to controls, whereas EGF excretion was decreased when expressed per kidney weight. Immunohistochemistry demonstrated that EGF and THP were colocalized in the thick ascending limbs of Henles loops and distal tubules in all five groups of rats. In conclusion, the EGF excretion appears to follow renal tissue mass and seems independent of urinary volume, whereas THP excretion is dependent mainly on urinary volume. This has implications for the use of EGF and/or THP excretion rates as an indicator for distal tubular function.

Processing of Epidermal Growth Factor in the Rat Submandibular Gland by Kallikrein-Like Enzymes

Epidermal growth factor (EGF) is synthesized as a precursor which is processed intracellularly to a 6 kDa EGF in the rat submandibular gland. This gland contains very high amounts of kallikrein-like enzymes, and the purpose of the present study was to examine whether any of five such enzymes, rK1, rK2, rK7, rK9 or rK10, can process the rat EGF precursor. Molecular weight forms of EGF, that were N- or C-terminally extended compared to submandibular gland EGF were obtained from rat urine. These extended forms of EGF were incubated with each of the enzymes for 24 h at 37 degrees C. Two enzymes, rK7 and rK10, were able to cleave N- and C-terminally extended EGF, releasing a form of EGF which eluted similarly to submandibular gland EGF upon gel filtration, and which was recognized both by antibodies against rat EGF and by the EGF receptor. One enzyme, rK1, cleaved C- but not N-terminally extended EGF. Neither rK2, nor rK9 cleaved the extended forms of EGF. In previous immunohistochemical studies rK1, rK7 and rK10 have all been demonstrated in the EGF containing cells of the rat submandibular gland. EGF and rK1 are also synthesized in the rat kidney but the present study demonstrated that EGF and rK1 are not colocalized in this organ. Based on the cleavage of the extended forms of rat EGF by rK1, rK7 and rK10 and on the fact that the enzymes are abundant and colocalized with EGF in the rat submandibular gland, we suggest that rK1, rK7 and rK10 can be involved in the processing of the EGF precursor in the rat submandibular gland.

Early urinary changes in Tamm-Horsfall protein and epidermal growth factor in diabetic children

Abstractu2002Both glomerular and tubular markers have been used to follow diabetic nephropathy. However, neither albumin nor proximal tubular markers have proven useful in prepubertal diabetes. Hence we studied two markers derived from the distal tubular cells, Tamm-Horsfall protein (THP) and epidermal growth factor (EGF). The urinary excretion of THP and EGF was examined in samples obtained during the first 20 days and 1 year after diagnosis of diabetes in children aged 4–15 years. Fourteen children without and 18 with ketonuria were examined, and 17 age-matched healthy children participated as controls. The excretion rate of EGF was increased at diagnosis, while that of THP was not. After 20 days of treatment the excretion of EGF had normalized, while the excretion of THP was decreased. Similar results were obtained after 1 year. In conclusion, in spite of good metabolic control a reduced excretion of THP persisted for at least 1 year after the diagnosis of diabetes. Whether the finding of reduced excretion of THP has any biological significance awaits further study.

Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.

Adrenergic Blockade in Diabetic and Uninephrectomized Rats: Effects on Renal Size and on Renal and Urinary Contents of Epidermal Growth Factor

The present study reports on the effects of adrenergic blocking agents on the renal growth and on the renal content and urinary excretion of epidermal growth factor (EGF) in streptozotocin-induced diabetic or uninephrectomized rats. Diabetic and uninephrectomized rats were allocated to groups treated with either saline or adrenergic antagonists and compared to controls and sham-operated controls, respectively. 24-hour urine samples were obtained on days 7, 14, and 21 and renal tissue samples on day 21. The 24-hour urinary excretion of EGF from controls and saline-treated diabetic rats was comparable. In adrenergic antagonist treated diabetic rats, it was reduced by at least 40% throughout the study period. Uninephrectomy caused a 50% reduction in the urinary excretion of EGF. This was not influenced by treatment with an adrenergic antagonist. After 3 weeks, saline-treated diabetic rats had an increase of 33% in kidney weight when compared to controls. The adrenergic antagonist treated diabetic groups had a significantly lower increase of 15%. Postnephrectomized renal growth was not affected by adrenergic antagonists. The total renal content of EGF was comparable in the saline-treated diabetic group and the control group, but was reduced by approximately 50% in the kidneys from the adrenergic antagonist treated diabetic groups. Renal EGF mRNA levels were also reduced in adrenergic antagonist treated diabetic rats. In contrast to diabetes, the renal growth following nephrectomy was not affected by adrenergic blocking agents. These results provide evidence for fundamental differences between diabetes-related renal growth and that observed in compensation to nephrectomy and suggest a connection between adrenergic activity, renal growth, and EGF in diabetes.

More Studies on Outcomes Using Biochemical Diagnostic Tests Are Needed: Findings from the Danish Society of Clinical Biochemistry

The results of biochemical tests often lead to diagnostic and therapeutic interventions, and the real value of a test can be assessed only by taking into account the subsequent health outcomes. The importance of outcomes studies, and the challenges in performing them, was reviewed by Bruns in 2001 (1), who argued that this type of study should be performed more frequently, and that such studies should be used to determine whether new tests should be implemented in clinical practice.nnTo investigate the extent to which this recommendation has been realized, a working group on evidence-based clinical biochemistry established by the Danish Society of Clinical Biochemistry undertook a pilot study to record the number and type of reports of diagnostic biochemical outcome studies published from January 2005 to January 2006 in 4 medical journals: Clinical Chemistry, Clinical Chemistry and Laboratory Medicine , Lancet and the …