Norbert Wild

A Combination of Serum Markers for the Early Detection of Colorectal Cancer

Purpose: Fecal occult blood testing is recommended as first-line screening to detect colorectal cancer (CRC). We evaluated markers and marker combinations in serum as an alternative to improve the detection of CRC. Experimental Design: Using penalized logistic regression, 6 markers were selected for evaluation in 1,027 samples (301 CRC patients, 143 patients with adenoma, 266 controls, 141 disease controls, and 176 patients with other cancer). The diagnostic performance of each marker and of marker combinations was assessed. Results: To detect CRC from serum samples, we tested 22 biomarkers. Six markers were selected for a marker combination, including the known tumor markers CEA (carcinoembryonic antigen) and CYFRA 21-1 as well as novel markers or markers that are less routinely used for the detection of CRC: ferritin, osteopontin (OPN), anti-p53, and seprase. CEA showed the best sensitivity at 95% specificity with 43.9%, followed by seprase (42.4%), CYFRA 21-1 (35.5%), OPN (30.2%), ferritin (23.9%), and anti-p53 (20.0%). A combination of these markers gave 69.6% sensitivity at 95% specificity and 58.7% at 98% specificity. Focusing on International Union against Cancer (UICC) stages 0–III reduced the sensitivity slightly to 68.0% and 53.3%, respectively. In a subcollective, with matched stool samples (75 CRC cases and 234 controls), the sensitivity of the marker combination was comparable with fecal immunochemical testing (FIT) with 82.4% and 68.9% versus 81.8% and 72.7% at 95% and 98% specificity, respectively. Conclusions: The performance of the serum marker combination is comparable with FIT. This provides a novel tool for CRC screening to trigger a follow-up colonoscopy for a final diagnosis. Clin Cancer Res; 16(24); 6111–21. ©2010 AACR.

Improved Diagnosis of Colorectal Cancer Using a Combination of Fecal Occult Blood and Novel Fecal Protein Markers

BACKGROUND & AIMS Annual testing for fecal occult blood is recommended as first-line screening for the detection of colorectal cancer (CRC), but is affected by limited sensitivity. We initiated a proteomics-based search for novel biomarkers to improve the sensitivity of detection of CRC in stool samples. METHODS Six markers, including immunologic fecal occult blood test (iFOBT), were evaluated in a collective of 551 samples (186 CRC, 113 advanced adenoma, and 252 control patients) to establish the diagnostic performance of each marker and marker combinations. RESULTS We tested the known stool markers hemoglobin (iFOBT), hemoglobin-haptoglobin, calprotectin, carcinoembryogenic antigen, and the novel fecal markers tissue inhibitor of metalloproteinase-1 (TIMP-1) and S100A12. The best diagnostic performance was found for S100A12 with an area under the curve of 0.95, followed by TIMP-1 (0.92), hemoglobin-haptoglobin (0.92), hemoglobin (0.91), calprotectin (0.90), and carcinoembryogenic antigen (0.66). By using Bayes logistic regression as a mathematic model, the highest sensitivity (88%) for the detection of CRC at 95% specificity was obtained with the marker pair S100A12 and hemoglobin-haptoglobin. Increasing the specificity to 98%, the combination of S100A12, hemoglobin-haptoglobin, and TIMP-1 resulted in a sensitivity of 82%, with the highest increase of sensitivity found in early tumor stages (international union against cancer stage I: 74% sensitivity vs 57% of the best single marker). CONCLUSIONS Depending on the specificity selected, a marker pair, S100A12 and hemoglobin-haptoglobin, or a triple combination including TIMP-1, allowed the detection of CRC at significantly higher rates than can be obtained with iFOBT alone.

Diagnosis of rheumatoid arthritis: multivariate analysis of biomarkers

Abstract Objective. To test if a combination of biomarkers can increase the classification power of autoantibodies to cyclic citrullinated peptides (anti-CCP) in the diagnosis of rheumatoid arthritis (RA) depending on the diagnostic situation. Methods. Biomarkers were subject to three inclusion/exclusion criteria (discrimination between RA patients and healthy blood donors, ability to identify anti-CCP-negative RA patients, specificity in a panel with major non-rheumatological diseases) before univariate ranking and multivariate analysis was carried out using a modelling panel (n=906). To enable the evaluation of the classification power in different diagnostic settings the disease controls (n=542) were weighted according to the admission rates in rheumatology clinics modelling a clinic panel or according to the relative prevalences of musculoskeletal disorders in the general population seen by general practitioners modelling a GP panel. Results. Out of 131 biomarkers considered originally, we evaluated 32 biomarkers in this study, of which only seven passed the three inclusion/exclusion criteria and were combined by multivariate analysis using four different mathematical models. In the modelled clinic panel, anti-CCP was the lead marker with a sensitivity of 75.8% and a specificity of 94.0%. Due to the lack in specificity of the markers other than anti-CCP in this diagnostic setting, any gain in sensitivity by any marker combination is off-set by a corresponding loss in specificity. In the modelled GP panel, the best marker combination of anti-CCP and interleukin (IL)-6 resulted in a sensitivity gain of 7.6% (85.9% vs. 78.3%) at a minor loss in specificity of 1.6% (90.3% vs. 91.9%) compared with anti-CCP as the best single marker. Conclusions. Depending on the composition of the sample panel, anti-CCP alone or anti-CCP in combination with IL-6 has the highest classification power for the diagnosis of established RA.

Towards a comprehensive proteome of normal and malignant human colon tissue by 2‐D‐LC‐ESI‐MS and 2‐DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.

Abstract 2738: Early detection of colorectal cancer applying a combination of serum markers

Background: Fecal occult blood testing (FOBT) is the recommended first line screening for the detection of colorectal cancer (CRC). To improve the detection of CRC we evaluated serum markers and combinations of serum markers as an alternative approach. Methods: Applying Lasso Regression, a specialized form of penalized logistic regression, we selected six markers for an evaluation in a collective of 857 patients including 301 CRC patients, 143 patients with adenoma, 266 healthy controls and 147 disease controls. For each marker and marker combination the performance was assessed. Results: We tested a total of 22 biomarkers for the detection of CRC from serum. Of these six markers were selected for a marker combination by Lasso Regression. Included were the well-known tumor markers CEA and CYFRA21-1 as well as novel markers or markers that are less routinely used for the detection of CRC: ferritin, osteopontin, anti-p53 and seprase. CEA showed the best sensitivity of all markers with 43.9 % at 95% specificity, followed by seprase (42.4%), CYFRA21-1 (35.5%), osteopontin (30.2%), ferritin (23.9%) and anti-p53 (20.0%). When these markers were combined a sensitivity of 72.3% was reached at a corresponding specificity of 95% and of 62.1% at 98% specificity. Focusing on more screening relevant stages, UICC stages 0-III, reduced the sensitivity slightly to 68.0% and 53.3%, respectively. In a sub-collective where matched stool samples were available (75 CRC cases and 234 controls) the sensitivity of the marker combination was comparable to fecal immunochemical testing (FIT) with 82.4% and 68.9% vs. 81.8% and 72.7% at 95% and 98% specificity, respectively. Conclusion: When six markers were combined to detect CRC from serum, the combination reached a performance that was comparable to FIT. This provides a novel tool for CRC screening to trigger a follow-up colonoscopy for a final diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2738.

From Research to Clinical Application Multi-Parameter Testing: Marker Panels for the Early Detection of Complex Diseases

From Research to Clinical Application Multi-Parameter Testing: Marker Panels for the Early Detection of Complex Diseases Multi Parameter Analysis can open novel diagnostic opportunities for the early diagnosis and screening of multimodal diseases like cancer. Single proteins have so far failed to describe such complex diseases. Being able to screen with a set of analytes is one promising way to overcome the present limitations. Various marker identification tools including proteomics approaches have been successfully applied to identify new screening markers for early detection of colorectal cancer (CRC). CRC is one of the most incident cancers worldwide and early detection is clearly a key factor in reducing mortality from CRC. Several screening methods are recommended, including colonoscopy, fecal occult blood testing (FOBT) and fecal DNA analysis. Of these annual stool testing with the guaiac based FOBT is most often applied, in spite of limitations such as low sensitivity and dietary influences. Though proce dures with improved performance eg. immunolo gical FOBT are available, a screening assay for CRC in serum that could easily be integrated in any health check-up would be highly welcome. A positive result of such a test would trigger a follow-up colonoscopy for an exact diagnosis. In this review we will cover aspects of marker identification strategies and describe a well structured marker validation process that is based on clinically characterized sample materials. Finally the value of analytical multi-parameter platforms enabling the combination of multi markers in routine diagnostics settings is outlined. An appropriate multi-parameter immunochemistry platform concept, currently developed under the working name »IMPACT« will be introduced. Od Istraživanja do Kliničke Primene: Paneli Markera za Ranu Detekciju Složenih Bolesti Analiza više parametara (multi-parameter analysis) može doneti nove dijagnostičke mogućnosti za ranu dijagnozu i testiranje multimodalnih bolesti poput raka. Pojedinačni proteini dosad nisu uspeli da opišu tako složene bolesti. Mogućnost testiranja pomoću seta analita najavljuje prevazilaženje postojećih ograničenja. Različite alatke za identifikaciju markera uključujući proteomske pristupe uspešno se primenjuju za identifikaciju novih markera za rano otkrivanje kolorektalnog kancera. Kolorektalni kancer je jedan od najčešćih kancera u celom svetu a rana detekcija je ključni faktor za smanjenje mortaliteta od te vrste raka. Preporučeno je nekoliko metoda za testiranje, uključujući kolonoskopiju, analizu okultnog krvarenja u stolici i analizu DNK u stolici. Najčešće se primenjuje godišnje testiranje stolice metodom gvajak zasnovano na analizi okultnog krvarenja u stolici, uprkos ograničenjima kao što su niska senzitivnost i uticaj ishrane. Mada postoje postupci s poboljšanim performansama, npr. imunološko testiranje okultnog krvarenja u stolici, skrining test na kolorektalni rak koji bi se lako integrisao u svaki zdravstveni pregled bio bi veoma korisan. Pozitivan rezultat takvog testa ukazao bi na potrebu za kolonoskopijom radi precizne dijagnoze. U ovom radu biće razmotreni aspekti strategija za identifikaciju markera i opisan valjano struktuiran postupak validacije markera zasnovan na klinički okarakterisanim uzorcima materijala. Najzad, biće naznačena vrednost analitičkih platformi s više parametara koje omogućuju kombinovanje multimarkera u rutinskoj dijagnostici. Biće predstavljen i koncept odgovarajuće imunohemijske platforme s više parametara koja se trenutno razvija pod radnim nazivom »IMPACT«.