Geneviève Choquet-Kastylevsky

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 μg/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 μl 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.

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.

Gell and Coombs's classification : is it still valid?

The Gell and Coombss classification divides drug allergies into four pathophysiological types, namely anaphylaxis (type I), antibody-mediated cytotoxic reactions (type II), immune complex-mediated reactions (type III), and delayed type hypersensitivity (type IV). Although this classification was proposed more than 30 years ago, it is still widely used. As only a limited number of drug allergies fit into this classification which does not include our current understanding of the immune response, its use is not recommended, particularly in the context of the preclinical safety evaluation of new therapeutic agents. In fact, three different situations can be identified, namely pseudo-allergic reactions, primarily antibody-mediated reactions and cell-mediated reactions, which could serve as a basis for modern and more adequate classifications

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 β, 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.

The current status of clinical proteomics and the use of MRM and MRM3 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 MRM3 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 p75NTR 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 p75NTR 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.

Endocrine and neurological adverse effects of the therapeutic interferons.

There is experimental evidence that the nervous central and the neuroendocrine systems can influence the immune system, which can in turn influence the brain activity. Endogenous cytokines are known to play a critical role in the pathophysiology of many diseases. The recently acquired experience on the adverse effects of therapeutic cytokines, particularly neurological and endocrine adverse effects, are further illustrative of these interferences. Interferons-alpha have been used in thousands of patients, so that the information accumulated with this group of closely related products is essential to delineate the potential and severity for non-immunological, but largely immune-mediated adverse effects to develop in patients treated with immuno-activating agents.

Responses of the Immune System to Injury

Three categories of immunotoxic effects are identified: direct immunotoxicity, hypersensitivity, and autoimmunity. Direct immunotoxicity consists of immunosuppression and immunostimulation. Total abrogation of the immune response (immunosuppression) results in more frequent, severe, and often atypical and relapsing infections and lymphomas. Immunostimulation is associated with febrile reactions, the induction/facilitation of autoimmune diseases and allergic reactions to unrelated allergens, and impaired hepatic drug biotransformation. Hypersensitivity is manifested by a variety of symptoms involving either antigen-specific or non-antigen-specific humoral and cellular adverse responses. Autoimmune reactions are divided into organ-specific and systemic reactions. Because of the involvement of many redundant mechanisms, it is difficult to predict responses of the immune system to a given immunotoxic injury. In laboratory animals, histologic but also functional changes are necessary to show evidence of and to predict such adverse responses.

Treatment of Drug-Induced Agranulocytosis with Haematopoietic Growth Factors

Although drug-induced agranulocytosis is infrequent, it is of concern as the mortality rate ranges from 6 to 10%. Since the approval of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), these drugs have been increasingly used in the management of drug-induced agranulocytosis. Unfortunately, most of the data regarding the use of these agents in patients with drug-induced agranulocytosis comes from case reports. In light of the low incidence of drug-induced agranulocytosis, the large variety of offending drugs with potentially different toxic mechanisms and the wide range of neutropenia duration among patients with agranulocytosis, randomised, double-blind studies are unlikely to be performed.Case reports provide promising results with a shortening in the duration of agranulocytosis, a possible reduction in the duration of hospitalisation and the fatality rate in patients treated with haematopoietic growth factors (HGF) compared with historical controls. A therapeutic effect is also suggested by reports of reductions in the neutrophil count after HGF discontinuation following an initial increase.The results of recent case series are less positive, with only a moderate, but usually not significant, reduction in the duration of neutropenia in patients treated with HGF, as compared with those receiving routine care. A Japanese study indicated that G-CSF was effective in patients with mild-to-moderate antithyroid drug-induced neutropenia, whereas no clear benefit was apparent in those with severe neutropenia.Several factors, for example, early recognition and improved management of individual cases with better supportive care, have contributed to a decrease in the overall mortality of drug-induced agranulocytosis. HGF are expected to further reduce mortality. Guidelines for the use of HGF in patients with febrile neutropenia, as established by the American Society of Clinical Oncology, are probably valuable for the management of drug-induced agranulocytosis. In accordance with these recommendations, the use of HGF may be recommended in patients with severe neutropenia and/or poor prognostic factors. Whether the absence of myeloid precursors or presence of promyelocytes or myelocytes in bone marrow examination represents optimal conditions for HGF treatment is still unknown. Most authors agree that treatment should be administered early in the course of the disease. An interesting approach, in which treatment decisions are based on the granulocyte count 4 hours after a single dose of G-CSF in patients with anthithyroid drug-associated neutropenia should be more extensively evaluated.

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.