Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease. METHODS: We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa. RESULTS: We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an 'interactome' with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6. CONCLUSIONS: Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Disturbances in peripheral blood B cell subpopulations in autoimmune patients.

A variety of cell surface markers are being used to identify B cell subpopulations in peripheral blood. Currently at least eight subpopulations have been identified. Analyses of healthy individuals indicate that in general the various B cell subpopulations exist in relatively similar ratios in unrelated individuals. It has been demonstrated that B lymphocyte homeostasis is disturbed during infection and autoimmune disease. In this review we compare the distribution of B cell subpopulations in the peripheral blood of patients with systemic lupus erythematosus, rheumatoid arthritis and primary Sjogren's syndrome with each other, and with healthy individuals. The different autoimmune diseases have distinct changes in the B cell subpopulations. Understanding the nature of these B subpopulation signatures will potentially impact understanding the mechanisms of disease, diagnosis and therapy.

Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing.

Hairy cell leukemia (HCL) derives from a mature B cell and expresses markers associated with activation. Analysis of immunoglobulin variable region genes has revealed somatic mutation in most cases, consistent with an origin from a cell that has encountered the germinal center. One unusual feature of hairy cells (HCs) is the frequent expression of multiple immunoglobulin heavy chain isotypes, with dominance of immunoglobulin (Ig)--G3, but only a single light chain type. The origin and clonal relationship of these isotype variants have been unclear. In order to probe the isotype switching status of HCL, RNA transcripts of V(H)DJ(H)--constant region sequences from 5 cases of typical HCL, all expressing multiple surface immunoglobulin isotypes, were analyzed. Tumor V(H)DJ(H)--C(mu) sequences were identified and found to be somatically mutated (range, 1.4% to 6.5%), with a low level of intraclonal heterogeneity. Additional immunoglobulin isotypes of identical V(H)DJ(H) sequence were also identified, including IgD (5 of 5), IgG3 (5 of 5), IgG1 (3 of 5), IgG2 (2 of 5), IgA1 (4 of 5), and IgA2 (1 of 5). Derivation of multiple isotypes from individual cells was demonstrated by analyzing transcripts in single sorted cells from one patient, with evidence for coexistence of isotype variants in 10 of 10 cells. These findings indicate that clonally related multiple isotypes coexist in single HCs, with individual isotypes presumably generated via RNA splicing. Production of IgG3 appears common, but IgG1, IgG2, IgA1, and IgA2 also arise, indicating a continuing influence of a directed process on the tumor clone. These HCs appear to be arrested at the point of isotype switch, where RNA processing may precede deletional recombination. (Blood. 2001;98:1174-1181)

Sequence analysis of V(4-34)-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE).

SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V(4-34) heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V(4-34)-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both V(H) and V(L) genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For V(H), all were derived from V(4-34) as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, V(L) (12 kappa and 3 lambda) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated V(H) sequences were associated with unmutated V(L) sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of V(H) ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the V(L)-J(L) junctions in 4/15. These findings provide insight into the V(4-34)-V(L) gene combinations used by B cells in patients with SLE which might have clinical relevance.

Phage surface expression for analysis of recognition sites of human autoantibodies: comparison of single chain Fv and Fab.

Human autoantibodies can mediate the pathology of autoimmune disease. To plan therapeutic intervention based on inhibition of binding to autoantigen, it is desirable to map the amino acid residues involved in recognition. Bacterial expression provides a simple and rapid system for production and mutagenesis of recombinant antibody fragments, but there are problems with reproducible folding and dispersity of soluble products. Expression at the surface of phage particles avoids both these problems, and allows variable region gene sequences to be expressed as functional monomers. To apply this technology, we have expressed a human anti-DNA monoclonal antibody, derived from a hybridoma, at the surface of the phage. The presence of the phage particle allowed highly sensitive detection of antibody activity by ELISA, so that minimal levels of expression were required. By measuring the amount of expressed antibody protein, it was possible to analyze the antibody activity/microgram. We expressed the antibody as FAB or single chain (sc) Fv, and found binding activities to be closely similar. The parental anti-DNA antibody utilizes the human V4-34 gene and displays the associated conformation-dependent 9G4 idiotope. Both the Fab and scFv expressed the idiotope, confirming similar molecular folding. This technology can be applied rapidly and efficiently to investigations of human autoantibody structure, with the option for expression as either Fab or scFv, and the only requirement being knowledge of VH and VL sequences.

Use of phage surface expression to analyze regions of human V4-34(VH4-21)-encoded IgG autoantibody required for recognition of DNA: no involvement of the 9G4 idiotope.

The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.

Tracking of the V4-34 (VH4-21) gene in human tonsil reveals clonal isotype switch events and a highly variable degree of somatic hypermutation.

The V4-34 (VH4-21) gene has been found to encode certain IgM autoantibodies, and is mandatory for pathological IgM anti-erythrocyte antibodies of I/i specificity. The gene is also commonly used by normal IgM-positive B lymphocytes, but its involvement in B cells which have undergone class switching to IgG or IgA is less clear. In order to track V4-34 gene usage and class switching events during a normal immune response, we have probed RNA in a limited area of human tonsil. Results indicate that the V4-34 gene undergoes class switching to IgG or IgA, with the progeny either remaining unmutated or containing large numbers of somatic mutations. Mutational patterns indicate possible 'hot spots', and some mutations appear deleterious. At the level of individual B cells, we have tracked a clonal isotype switch event from IgM to IgA, with each retaining close to germ-line configuration. In addition, we have followed a clonal switch from a mutated IgM to IgG, with no further accumulation of somatic mutations. These data indicate that the V4-34 gene is involved in a maturing immune response, and that the routes to production of IgG or IgA antibodies are various.

Analysis of VH genes used by neoplastic B cells in endemic Burkitt's lymphoma shows somatic hypermutation and intraclonal heterogeneity.

Tumor cell lines from six typical cases of endemic Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig VH genes. The neoplastic cells all had a t(8;14) (q24;q32) translocation involving the c-myc protooncogene. The VH genes were derived from VH1, VH3 and VH4, and both the IgM-positive (four cases) and IgG-positive (two cases) were extensively mutated from germline sequence. In two cases, early and late passage tumor cells were available, and the VH nucleotide sequences were identical, indicating that mutations had not accumulated in vitro. In a further case, there was evidence of sequence heterogeneity, which appeared to have been generated in vivo, indicating that the tumor cell VH gene was able to undergo posttranslocation somatic hypermutation. Analysis of the relatively nonpolymorphic VH4 genes for the pattern of replacement or silent mutations did not show a role for antigen selection in the expressed sequences.

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus.

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.

Radioimmunoassayable insulin-like growth factor-I in human breast cyst fluid.

insulin-like growth factor-I (IGF-I) was measured in breast cyst fluid and serum collected at the same time. In addition, epidermal growth factor (EGF) was measured in breast cyst fluid. The radioimmunoassay inhibition curve of cystic IGF-I was parallel to that of authentic IFG-I. Cystic immunoreactive IGF-I had an elution pattern similar to IGF-I from Sep-Pak C18 silica columns and from gel-filtration chromatography using Sephacryl 200. The material from Sephacryl 200 gave a radioimmunoassay curve which was parallel to IGF-I. The median (range) amount of IGF-I in cyst fluid was 9.3 ng/ml (0.8-33.3 ng/ml) and was 5-10% of that found in serum, although there was no significant correlation between the two levels. The median (range) level of cystic EGF was 416 ng/ml (14-1640 ng/ml). There was a weak negative correlation between cystic EGF and cystic IGF-I (P less than 0.01). Some women presented with multiple cysts and for these cysts there was a high degree of concordance of levels for both IGF-I or EGF. Unlike serum there was little or no protein present in cyst fluid which bound IGF-I.

Measurement of human corticosteroid binding globulin by enzyme-linked immunoassay.

Thirteen monoclonal antibodies have been raised against corticosteroid binding globulin (CBG). From four of those with highest affinity for the antigen, two were selected for development of a sandwich enzyme-linked immunoassay (ELISA). The sensitivity of the assay was such that 0.7 fmol CBG could be detected. Levels of the binding protein in men (740 +/- 67 nmol/l) and women (690 +/- 103 nmol/l) were not significantly different, while those found during the third trimester of pregnancy (1500 +/- 423 nmol/l) were approximately twice these levels. CBG denatured by heating to 60 degrees C could not be detected by the ELISA.

Urinary epidermal growth factor excretion and breast cancer risk.

The amount of urinary epidermal growth factor (EGF) excreted was determined in 350 normal women of whom 37 subsequently developed breast cancer. These were a group of women selected on a case-control basis from 5000 volunteers who had participated in a prospective epidemiological study. Urinary EGF excretion was not correlated with known risk factors such as age at menarche or menopause, age at first or last full-term child or parity. Neither was it associated with day or length of menstrual cycle, breast mammographic parenchymal pattern or the blood concentration of prolactin, dehydroepiandrosterone or its sulphate ester. Univariate analysis indicated that the amount of urinary EGF was significantly correlated with urinary creatinine (P less than 0.001), age (P less than 0.001), urinary androsterone (P less than 0.02) or aetiocholanolone (P less than 0.02), height (P less than 0.05) and weight (P less than 0.05). However, multivariate analysis showed that the amount of urinary EGF was correlated only with creatinine excretion (P less than 0.001) and age (P less than 0.001) and that the significance of the other correlations were probably due to the confounding influence of creatinine.