Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability.

OBJECTIVES: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. DESIGN AND METHODS: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31). RESULTS: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month. CONCLUSIONS: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.

Characterization of three amino-functionalized surfaces and evaluation of antibody immobilization for the multiplex detection of tumor markers involved in colorectal cancer.

Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.

The current status of clinical proteomics and the use of MRM and MRM(3) for biomarker validation.

The transfer of biomarkers from the discovery field to clinical use is still, despite progress, on a road filled with pitfalls. Since the emergence of proteomics, thousands of putative biomarkers have been published, often with overlapping diagnostic capacities. The strengthening of the robustness of discovery technologies, particularly in mass spectrometry, has been followed by intense discussions on establishing well-defined evaluation procedures for the identified targets to ultimately allow the clinical validation and then the clinical use of some of these biomarkers. Some of the obstacles to the evaluation process have been the lack of the availability of quick and easy-to-develop, easy-to-use, robust, specific and sensitive alternative quantitative methods when immunoaffinity-based tests are unavailable. Multiple reaction monitoring (MRM; also called selected reaction monitoring) is currently proving its capabilities as a complementary or alternative technique to ELISA for large biomarker panel evaluation. Here, we present how MRM(3) can overcome the lack of specificity and sensitivity often encountered by MRM when tracking minor proteins diluted by complex biological matrices.

Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein.

The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer.

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit ß, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5-15%) and sensitive (0.3 ng·mL(-1)) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.

Clinical quantitation of prostate-specific antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) coupling and correlation with ELISA tests.

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low microg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 microl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.

Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2-DE.

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Cancer immunomics using autoantibody signatures for biomarker discovery.

The increased incidence of autoantibodies in malignancies has been described since the 1970s. Thus the ability to determine molecular fingerprinting of autoantibodies (antibody signatures) may provide useful clinical diagnostic and prognostic information. This review describes the use of several proteomics approaches for the identification of antigens recognized by these autoantibodies. Serological proteome analysis combines separation of tumor cell proteins on two-dimensional gel electrophoresis gels, Western blotting with sera of patients and healthy subjects, and identification of the detected antigens by MS. Alternatively multiple affinity protein profiling combines isolation of the antigens recognized by patient antibodies by two-dimensional immunoaffinity chromatography and identification by MS/MS. The use and limitations of reverse phase protein microarrays for testing patient serum containing autoantibodies are also considered. Lastly the most important difficulty of any proteomically identified autoantibody signature is validation in patient cohorts or clinical samples.

SELDI-TOF MS analysis of the Cardiac Troponin I forms present in plasma from patients with myocardial infarction.

The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins.

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.

Characterization and enumeration of cells secreting tumor markers in the peripheral blood of breast cancer patients.

In the process of metastasis, malignant cells are released from the primary tumor and migrate to specific organs via the lymphatic and blood circulation systems. These circulating tumor cells have been characterized by immunochemistry, the reverse transcription-polymerase chain reaction, and flow cytometry. Using the MCF-7 breast cancer cell line, we have developed a two-color ELISPOT assay to detect cells secreting cathepsin D protease and MUC1 glycoprotein, markers associated with the risk of metastases in breast cancer. The threshold of detection of this ELISPOT assay was one cathepsin D- or MUC1-secreting MCF7 cell per 5 ml of control blood. In 16 patients with breast carcinoma metastases, 1 to 1940 cathepsin D- or MUC1-secreting cells per 2x10(7) PBMC were enumerated, whereas none were found in 11 controls. Moreover, in six patients 6-60% of MUC1-secreting cells also expressed the CXCR4 chemokine receptor, which is involved in the homing of metastatic breast cancer cells. The ELISPOT assay described here allowed us to enumerate cathepsin D- and/or MUC1-secreting cells in the MCF-7 cell line and in the peripheral blood of patients with disseminated breast cancer. The combination of the ELISPOT assay and CXCR4-positive cell sorting identified subsets of MUC1-secreting cells in the peripheral blood of these patients.

Popliteal lymph node responses to acetone and ethanol differ from those induced by streptozotocin.

The popliteal lymph node (PLN) assay was proposed to detect the potential of immunotoxicants for inducing systemic autoimmune-like reactions, but also xenobiotics that are sensitizing or exert immunostimulatory properties. Results on over 100 chemicals, mostly pharmaceuticals, are available with the PLN assay and show many correlations between rodent data and the clinical experience. A major issue is that the mechanisms involved have not been fully elucidated. In order to provide mechanistic clues to improve the predictability of the PLN assay, the effects of streptozotocin (STZ) were compared to those of ethanol and acetone in normal C57Bl/6 mice as well as mice depleted in CD4+ or CD8+ T-cells by treatment with specific monoclonal antibodies. STZ, ethanol and acetone gave similar positive responses in normal mice. Neither CD4+ nor CD8+ T-cell depletion influenced the PLN responses to ethanol or acetone, whereas CD8+ in contrast to CD4+ T-cell depletion abolished the response to STZ. There was an increase in the production of IL-6 and IFN-gamma mRNAs measured by RT-PCR in STZ-, but not in ethanol- or acetone-treated normal mice. The production of TNFalpha, IL-1alpha, IL-1beta, IL-2R and IL-12 mRNAs was increased whatever the treatment, but increases were 2- to 3-fold greater after STZ than ethanol or acetone. These results suggest that PLN responses to primary irritants such as ethanol and acetone essentially reflect non-specific inflammation, whereas PLN responses to an autoimmunogenic compound such as STZ involve CD8+ T lymphocytes and the production of IFN-gamma and IL-6. These findings may prove useful to improve the predictability of the PLN assay.

Allergic adverse reactions to sulfonamides.

Antimicrobial sulfonamides were the first antimicrobial agents used effectively to treat infectious diseases. However, because they may cause severe adverse drug reactions (ADRs) and because more effective agents have since been developed, sulfonamides now are used for only a few indications in specific groups, such as AIDS patients. Skin reactions, from benign rash to potentially lethal toxidermias, are the most frequent ADRs to sulfonamides. Other major ADRs include acute liver injury, pulmonary reactions, and blood dyscrasias. Although the mechanisms involved have not been fully elucidated, reactive metabolites appear to play a pivotal role. The hydroxylamine and nitroso metabolites of sulfamethoxazole, the most frequently used sulfonamide today, can bind covalently to proteins because of their chemical reactivity, resulting in the induction of specific adverse immune responses. Therefore, changes in the activity of metabolic and detoxification pathways are associated with a greater risk for developing allergic reactions to sulfonamides. Allergies to sulfonamides, particularly sulfamethoxazole (often used in combination with trimethoprim as co-trimoxazole), are more frequent in AIDS patients, but the reason for this increased risk is not fully understood. No valid tools are available to predict which patients have a greater risk for developing allergies to sulfonamides. Diagnosis is essential to avoid a possible evolution toward severe reactions and readministration of the offending drug. In patients who absolutely require further treatment, successful desensitization may be achieved.