Purification of human chorionic gonadotropin from pregnancy urine by immunoaffinity chromatography using a monoclonal antibody anti-beta chain hCG

Human chorionic gonadotropin (hCG) is found in pregnancy urine and in urine from some cancer patients. It is found in a variety of forms, whose concentrations have clinical importance. A highly active preparation of hCG from pregnancy urine has been obtained by a simple method of immunoaffinity chromatography using a monoclonal antibody (MAb) against beta-subunit hCG. The recuperation of hCG from urine extract by this procedure was 84.18%, with a purification factor of 19.78. The specificity activity of the obtained hCG was 9705.36 IU/mg. The preparation appeared homogeneous by SDS-PAGE, analytical gel filtration, and double immunodiffusion. The molecular mass was 40 kDa by gel filtration on Superose 12 HR. Molecular masses estimated by electrophoresis were 14.6 and 23.5 kDa for the alpha and beta subunits, respectively.     

Isolation and characterization of 5T4, a tumour-associated antigen

The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin- and immunoaffinity chromatography and gel filtration giving up to 10,000-fold purification with 70% yield. The antigen is carried by non-associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS-PAGE and a neutral pl. Removal of N-linked sugars by N-glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O-linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N-linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.     

Characterisation of a humanised bispecific monoclonal antibody for cancer therapy.

A humanised bispecific monoclonal antibody (bsMAb) with binding specificity for carcinoembryonic antigen (CEA) on one arm and a radiolabelled chelate (DTPA-90Y) on the other arm was generated by consecutively transfecting the humanised genes of an anti-CEA MAb and the chimerised genes of an anti-chelate MAb into eucaryotic BHK cells using the calcium-phosphate coprecipitation technique. The antibodies secreted were of IgG3 isotype with a shortened hinge region (delta gamma 3) and light chains. Double transfectomas were screened for the secretion of bsMAbs using a double determinant enzyme-linked immunosorbent assay (ELISA) based on solid phase attached HSA-benzyl-DTPA and an anti-idiotypic MAb selective for the CEA-specific arm. After purification on two immunoaffinity chromatography columns, the humanised bsMAbs were characterised by SDS-PAGE and a quantitative binding assay in antigen excess. The purification procedure resulted in 95% reactive bispecific MAb. This humanised bsMAb may be employed in two phase radioimmunotherapy, binding to the tumour via the anti-CEA arm and localising a radiolabelled chelate with the other arm, without inducing a strong immune response observed sometimes with murine MAbs.     

抗前胃泌素释放肽单克隆抗体的纯化及体外对小细胞肺癌的活性

目的探讨亲和层析法纯化抗前胃泌素释放肽（PGRP）单克隆抗体（MAb）的效果,为该法纯化其他抗体提供数据依据。方法采用蛋白A-琼脂糖亲和层析法纯化含有MAb的腹腔积液,并用SDS—PAGE和酶联免疫吸附（ELISA）法分别检测其纯度和效价,用流式细胞术和免疫组织化学法检测其对小细胞肺癌细胞株NCI—H446的组织切片的生物功能。结果亲和层析法纯化前小鼠腹腔积液蛋白质质量浓度平均为23．62mg／ml;纯化前、后蛋白总量分别为148．79和146．67mg,回收率为98．58％。腹腔积液中MAb质量浓度平均为5．21mg／ml;纯化的MAb纯度达95％以上。纯化后抗体免疫学活性均高于纯化前,提高了6．90～15．40倍。结论蛋白A-琼脂糖亲和层析法可快速、高效地从小鼠腹腔积液中纯化抗PGRPMAb,且抗体具有较高的纯度,免疫学活性明显提高,能够选择性的和小细胞肺癌细胞的PGRP结合。     

Monoclonal antibody PD41 recognizes an antigen restricted to prostate adenocarcinomas.

A monoclonal antibody (MAb) designated PD41 (IgG1k) was generated by hyperimmunizing BALB/c mice with a membrane preparation prepared from a moderately to poorly differentiated prostate carcinoma surgical specimen. The immunohistochemical reactivity of MAb PD41 was shown to be highly restricted to the ductal epithelia and secretions of prostate adenocarcinoma tissues. Sixty-five % of the prostate tumor specimens were stained with MAb PD41, whereas no staining of the fetal or benign prostate specimens was observed. PD41 reacted minimally with normal prostate tissues, with less than 1% of the epithelial cells staining. This MAb did not react with nonprostate carcinomas or to a variety of normal human tissues. Using both radioimmunoassay and immunofluorescent procedures, several cultured human tumor cell lines, human blood cells, and purified antigens to prostate-specific antigen and prostatic acid phosphatase also were found not to express the PD41 antigen. MAb PD41 also was shown to bind to the target antigen present in seminal plasma obtained from prostate carcinoma patients but not to seminal plasma from normal donors. Immunoblots of gel-separated components of prostate carcinoma tissue extracts indicate that the molecular weight of the proteins carrying the PD41 antigenic determinant can differ among individual tumors, ranging from Mr 90,000 to greater than 400,000. However, in seminal plasma from prostate cancer patients, the predominant component recognized by PD41 is the diffuse Mr greater than 400,000 band. It appears that this monoclonal antibody may recognize a prostate carcinoma-associated mucin-like antigen, which is preferentially expressed on prostate carcinomas, and therefore, may be a useful marker to distinguish benign prostate hyperplasia from prostate carcinoma.     

Polymorphic epithelial mucin from the sera of advanced breast cancer patients--isolation and partial characterisation.

The anti-breast carcinoma monoclonal antibody (MAb), NCRC-11 defines a polymorphic epithelial mucin (PEM) which is elevated in the circulation of advanced breast carcinoma patients. Here we describe the purification and partial characterisation of this component from patients' sera and its use in the production of a second generation MAb, C568 (IgM). Pooled sera was fractionated by immunoaffinity and size-exclusion chromatography and the purity of preparations assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Serum-derived PEM shows a similar pattern of electrophoretic mobility to PEM isolated from primary breast tumour tissue and migrates as several bands in 4% SDS polyacrylamide gels (Mr greater than 400,000). The epitope expression of PEMs isolated from either source is also similar, with both bearing topographically distinct determinants for several anti-mucin MAbs. The immunoreactivities of antibodies C568 and NCRC-11 were unaffected by boiling, reduction and alkylation, or by enzyme desialylation of PEM. Periodate oxidation and proteolytic digestion have suggested that the antigenic determinant for C568 is carbohydrate in nature whilst that of NCRC-11 is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive beta-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-1.     

Purification and characterization of carcinoembryonic antigen from GW-39, a xenografted human colonic tumor system

Carcinoembryonic antigen (CEA) was purified from GW-39 human tumor xenografts in hamsters by immunoaffinity chromatography. Binding of the antigen to immobilized monoclonal antibody provided a high degree of purification of CEA in a single step. A recovery of 79% and a 750-fold purification were obtained. The purified CEA has a molecular size of 180 kilodaltons, an isoelectric point of 4.4, and a specific activity of 0.94. About 73% of the radiolabeled GW-39 CEA reacted with goat anti-CEA serum. The amino acid and carbohydrate compositions of the GW-39 CEA were comparable to those of CEA preparations from other sources.     

Isolation of plasma-membrane components from cultured human pancreatic cancer cells by immuno-affinity chromatography of anti-beta 2M sepharose 6MB.

Human pancreatic exocrine adenocarcinoma cells established in tissue culture expressed both HLA and beta 2-microglobulin (beta 2M). Plasma-membrane components of this pancreatic cancer cell line were purified from plasma membrane fractions enriched by sucrose density-gradient centrifugation, using immunoaffinity chromatography on immobilized anti-human beta 2M antibody. Both rabbit and mouse monoclonal anti-beta 2M IgG were used, with a 20--25-fold overall purification of 5'-nucleotidase. The method was applicable to 5 x 10(7) cells and permitted the solubilization of membranes retained on the column, with the selective desorption of components not associated with beta 2M before the subsequent elution at pH 3 of beta 2M-associated macromolecules. The acid eluate contained one major and two minor bands in the 40--45,000 mol.-wt range with two additional enriched components of 18,000 and 22,000 dalton. A major carbohydrate-containing component of high mol. wt was also found to be associated with the pancreatic cancer-cell plasma membrane.     

Selective DNA binding of the human cellular myb protein isolated by immunoaffinity chromatography using a monoclonal antibody

The bacterially expressed v-myb protein served as antigen for the isolation of several monoclonal antibodies, one of which recognized the human cellular myb protein (p75hu-c-myb) indicating a conserved epitope. The epitope was mapped to amino acid positions 208-232 by the use of several bacterially expressed v-myb proteins with various deletions. Furthermore, a synthetic oligopeptide which had been selected on the basis of its hydrophilicity by computer analysis of the v-myb oncogene (amino acids 213-231) blocked the action of this monoclonal antibody, indicating the immunological significance of this region. The monoclonal antibody allowed efficient purification of the p75hu-c-myb protein by immunoaffinity chromatography. The purified protein binds to double-stranded DNA in vitro in a filter-binding assay. Since the monoclonal antibody does not interfere with DNA binding it allowed analysis of DNA-protein interaction in a modified McKay assay using the purified p75hu-c-myb protein. Specific binding was observed predominantly to one of 12 lambda DNA fragments in vitro in the presence of high molar excess of competing co-polymer poly [d(I:C)]. Enhancer/promoter-like sequences of SV40 were not preferentially recognized.     

Enrichment of murine marrow cells for progenitors of multilineage hemopoietic colonies.

We attempted to develop a purification method for cell cycle-dormant murine multipotential progenitors that is simple and has a high recovery. Multilineage colony formation from mice that had been treated with 150 mg/kg 5-fluorouracil was assayed in cultures containing interleukin-3, interleukin-6, and erythropoietin. The cells with density 1.0631-1.0770 g/cm3 were approximately 40-fold enriched for multipotential progenitors. Negative immunomagnetic selection with a panel of lineage-specific monoclonal antibodies including anti-B220, anti-Gr-1, anti-Mac-1, anti-L3T4, and anti-Lyt-2 resulted in a cumulative 150-fold enrichment. When the density-separated and lineage-negative cells were further enriched by fluorescence-activated cell sorting with monoclonal antibody D7 (anti-Ly-6A/E), total enrichment averaged 800-fold and recovery was 17%. The method described here provides a relatively simple technique for the isolation of the multipotential progenitors and should be useful for the studies of regulation of hemopoiesis.     

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification

The preparation and characteristics of compound-specific andgroup-specific antibodies against 7-alkylguanines (7-alkGua)are described. A compound-specific antibody against 7-methylguaninewas prepared using a hapten bound to carrier protein throughthe N² position. In a competitive enzyme-linked immunosorbantassay (ELISA) 7-methyl-guanine (7-MeGua) showed 50% inhibition(I_50% at 10 pmol/ well at room temperature, but the inhibitionwas found to be 40 times better at 4°C (I_50% at 250 fmol/well).When the antibody was bound to protein A-Sepharose CL4B 7-MeGuawas retained in immunoaffinity columns. A group-specific antibodyto 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua)bound to carrier protein via the carboxyl group. In a competitiveELISA, this antibody cross- reacted well with 7-CEGua, 7-ethylguanine(7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine(7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinitycolumns prepared from this antibody retained a number of 7-alkGuaof diverse structure. 7-EtGua in calf thymus DNA treated withdiethyl sulphate and ethylnitrosourea was isolated by immunoaffinitypurification and quantified by HPLC-fluorescence. These resultsillustrate the potential of immunoaffinity purification forboth individual DNA adducts and groups of adducts.     

A pilot trial of CTLA-4 blockade with human anti-CTLA-4 in patients with hormone-refractory prostate cancer.

PURPOSE: Blockade of the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) augments and prolongs T-cell responses and is a strategy to elicit antitumor immunity. The objectives of this pilot study were to establish the pharmacokinetic and safety profile for a single dose of 3 mg/kg of the anti-CTLA-4 antibody Ipilimumab (MDX-010, BMS-734016) and to assess if this therapy resulted in prostate-specific antigen (PSA) modulation and the development of polyclonal T-cell activation and/or clinical autoimmunity in patients with hormone-refractory prostate cancer treated with Ipilimumab. EXPERIMENTAL DESIGN: Patients with metastatic hormone-refractory prostate cancer received a single 3 mg/kg i.v. dose of Ipilimumab. Serologic measures of autoimmunity were obtained, and T-cell activation was evaluated by flow cytometry. Pharmacokinetic sampling of plasma for MDX-CTLA-4, PSA measurement, and diagnostic imaging were also undertaken. RESULTS: Fourteen patients were treated: 12 patients received a single dose of Ipilimumab, and 2 patients were re-treated with a second dose upon PSA progression. Two patients showed PSA declines of > or =50%. Treatment was well tolerated with clinical autoimmunity limited to one patient who developed grade 3 rash/pruritis requiring systemic corticosteroids. The mean +/- SD Ipilimumab terminal elimination half-life was 12.5 +/- 5.3 days. CONCLUSIONS: A single dose of 3 mg/kg Ipilimumab, an anti-CTLA-4 antibody, given to patients with prostate cancer is safe and does not result in significant clinical autoimmunity. PSA-modulating effects observed warrant further investigation.     

Monoclonal antibodies and rabbit antisera recognizing 4-aminobiphenyl--DNA adducts and application to immunoaffinity chromatography.

Monoclonal antibodies and rabbit antisera were produced that recognized 4-aminobiphenyl, its major DNA adducts and other metabolites. The antigens used to raise these antibodies were synthesized by coupling the aromatic amine to protein through a diazotization reaction. The goal of this immunization strategy was to induce antibodies that also cross-reacted with most 4-aminobiphenyl-derived metabolites. A total of 20 mice and four rabbits were immunized and every animal produced a strong immune response for 4-aminobiphenyl and its derivatives. Two IgG1 monoclonal antibodies, 3D6 and 2E11, were isolated from two different mouse spleen cell fusions. One of the monoclonal antibodies, 3D6, had a high recognition for the three major 4-aminobiphenyl-DNA adducts: N-(deoxyguanosine-8-yl)-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosine-N2-yl)-4-aminobiphenyl, with affinity constants between 2 and 4 x 10(9) l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 10(9) l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobiphenyl-DNA adduct-specific antibodies. Preparative immunoaffinity resins were made for each monoclonal antibody. These resins quantitatively bound 500 ng each [3H]N-acetyl-aminobiphenyl, [3H]N-aminobiphenyl and [3H]N-(deoxyguanosine-8-yl)-4-aminobiphenyl. Preliminary experiments were performed to test the applicability of the preparative monoclonal antibody immunoaffinity column to isolate [3H]4-aminobiphenyl-derived metabolites in dosed rat and dog urine. About 70% of the radioactivity in rat or dog urine could be bound to the immunoaffinity columns. The combined immunoaffinity column/HPLC analysis of the dog urine led to the identification of a novel urinary metabolite, N-formyl-aminobiphenyl. HPLC analysis of a rat urine sample tentatively found 4-aminobiphenyl, N-acetyl-4-aminobiphenyl and N-formyl-4-aminobiphenyl by co-chromatography, and these compounds accounted for 20, 6.8 and 6.5% of the total radioactivity in the chromatogram respectively. Taken together, these data show that these 4-aminobiphenyl-specific monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.     

Identification of prostate specific membrane antigen (PSMA) as the target of monoclonal antibody 107-1A4 by proteinchip; array, surface-enhanced laser desorption/ionization (SELDI) technology

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. The identity of the antigen has been confirmed by Western blots using MAbs of known specificity. MAb 107-1A4 is not reactive on Western blots. The conformational epitope for 107-1A4 is on the extracellular domain of PSMA. In competition studies, the binding of MAb 107-1A4 to LNCaP cells is inhibited by itself but not by any other of several other anti-PSMA MAbs, suggesting that the epitope may be unique. These results suggest that 107-1A4 is reactive to a conformational epitope in the external domain of PSMA that is unique among the panel of anti-PSMA MAbs in this study. Furthermore this work demonstrates the ability of mass spectroscopy to elucidate antibody-ligand interaction.     

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.     

Individual variations of total and percent free serum prostatic specific antigen: could they change the indication of prostatic biopsy?

The aim of this study was to analyse the individual variations of total and percent free serum prostatic specific antigen (PSA) and to evaluate whether they could change the indication for prostatic biopsy. Prostatic needle biopsy was indicated in 63 patients with serum PSA between 4.0 and 10 ng/ml. A new determination of total and free PSA was done before the biopsy procedure. The time between the determinations ranged from 29 to 59 days. The total and free serum PSA determinations were performed by a double monoclonal antibody radioimmunoassay Tandem and Tandem free PSA. The median coefficient of variation for serum PSA was 12.9 in cancer free patients and 18.8 when cancer was detected, it was 32.6 and 42.2 respectively for percent free serum PSA. A 22.8% rate of discrepancy between the determinations was found when prostatic biopsy was indicated only by percent free PSA lower than 25. Sensitivity ranged from 93.3% to 100, and reduction of unnecessary biopsies between 15.2 and 21.8%. We conclude that individual variations in total and percent free serum PSA could have clinical implications because of the possibility that it changes the indication for a prostatic biopsy.     

Purification and composition of a novel gastrointestinal tumor-associated glycoprotein expressing sialylated lacto-N-fucopentaose II (CA 19-9)

Monoclonal antibody 1116NS 19-9 (Mab 19-9) exhibits selective reactivity with human gastrointestinal carcinomas and recognizes a carbohydrate determinant (CA 19-9) defined as a sialylated lacto-N-fucopentaose II. A scheme was devised for the purification of a human gastrointestinal tumor-associated glycoprotein antigen expressing CA 19-9 from colorectal carcinoma cell line SW1116 culture media in high yield. The key steps in the purification were immunoaffinity column chromatography with Mab 19-9 followed by reduction and alkylation of the specifically bound proteins in the presence of 6 M guanidine hydrochloride and a second Mab 19-9 immunoaffinity fractionation. The purified CA 19-9 containing glycoprotein ran as a single band on sodium dodecyl sulfate-polyacrylamide gradient gels with an apparent molecular mass of 210 kilodaltons. In the absence of detergents, this purified glycoprotein apparently reassociated to form aggregates of 600-2000 kilodaltons molecular mass as determined by size-exclusion chromatography. Amino acid analysis of CA 19-9 containing glycoprotein revealed that serine, threonine, and proline together accounted for greater than 35% of the amino acid residues, consistent with a mucin-like structure for the protein. Carbohydrate compositional analysis, however, was in contrast to a typical mucin with a fucose:mannose:galactose:N-acetylgalactosamine: N-acetylglucosamine:N-acetylneuraminic acid molar ratio of 4:1:12:2.5:5:5. The presence of both N-acetylgalactosamine and mannose suggested that both O- and N-linked oligosaccharides may exist on CA 19-9 containing glycoprotein. Protein and carbohydrate analyses indicated that this novel tumor-associated glycoprotein was 85% carbohydrate by weight. This purification procedure may be applicable to the isolation of other epithelial tumor-associated antigens.     

胃癌单克隆抗体MGcl相应抗原的初步研究

本文报告一株鼠抗人胃癌单克隆抗体MGcl相应抗原的纯化及分析。MGcl经50％饱和硫酸铵盐析,DEAE—52纤维素柱层析后,获得纯化的IgG56,31mg与2g溴化氰活化的sepharose 4B偶联,制备免疫吸附剂。用MGcl免疫亲和层析的方法从胃癌组织中纯化出MGcl相应抗原,经蛋白酶处理,高碘酸氧化,电泳脂蛋白袭色,SDS—PAGE及Immunoblotting等实验分析证实MGcl相应抗原是一种低分子量的糖蛋白,分子量为72Kd和52kd。     

Comparison of human prostate specific glandular kallikrein 2 and prostate specific antigen gene expression in prostate with gene amplification and overexpression of prostate specific glandular kallikrein 2 in tumor tissue.

BACKGROUND: There is a need for specific markers that can indicate the different stages of prostate carcinoma. There is ongoing speculation concerning the value of prostate specific glandular kallikrein (hK2) in this regard. METHODS: The expression levels of both hK2 and human prostate specific antigen (hPSA) were compared at the mRNA and protein levels by using in situ hybridization and immunohistochemistry techniques in tissue specimens from patients with benign prostatic hyperplasia and malignant prostate carcinoma. The respective gene copy numbers were analyzed by a competitively differential polymerase chain reaction-based method. RESULTS: In situ hybridization revealed that hK2 was expressed at significantly higher levels in malignant prostate tissue compared with benign prostate tissue (P < 0.0005), whereas hPSA expression levels were the reverse (P = 0.06). In benign tissue, the mean level of hK2 mRNA was 82% of the respective value of hPSA (P < 0.003), whereas, in tumor tissue, the mean hK2 expression level was 21% higher than that of hPSA (P < 0.01). The results at the protein level supported the mRNA findings: hPSA expression was lower in malignant tissues compared with benign tissues (17 of 20 specimens), whereas an increase in hK2 expression was detected in 17 of 19 specimens. The authors report that the hK2 gene (hKLK2) was amplified in prostate carcinoma tissue, whereas the hPSA gene was not. There was a correlation between hPSA and hK2 mRNA levels in both benign tissue (correlation coefficient [r] = 0.735; P < 0.01) and malignant tissue (r = 0.767; P < 0.01). CONCLUSIONS: Gene amplification of hKLK2 may be one of the factors leading to higher expression of hK2 in prostate carcinoma. The correlation between hK2 and hPSA expression levels indicates coordinated expression of the genes in both normal and abnormal prostate gland. The results suggest the potential value of hK2 in the diagnosis of prostate carcinoma through mRNA analyses and gene amplification.     

Introducing a prognostic score for pretherapeutic assessment of seminal vesicle invasion in patients with clinically localized prostate cancer.

PURPOSE: To identify prostate cancer patients who will have the most likely benefit from sparing the seminal vesicles during 3D conformal radiation therapy. METHODS AND MATERIALS: From 1988 to 2001, 532 patients underwent radical prostatectomy for clinically localized prostate cancer. Primary endpoint was the pathological evidence of seminal vesicle invasion. Variables for univariate and multivariate analyses were age, prostate weight, clinical stage, PSA level, Gleason score, number and site of positive prostate sextant biopsies. Multivariate logistic regression with backward stepwise variable selection was used to identify a set of independent predictors of seminal vesicle invasion, and the variable selection procedure was validated by non-parametric bootstrap. RESULTS: Seminal vesicle invasion was reported in 14% of the cases. In univariate analysis, all variables except age and prostate weight were predictors of seminal vesicle invasion. In multivariate analysis, only the number of positive biopsies (P<0.0001), Gleason score (P<0.007) and PSA (P<0.0001) were predictors for seminal vesicles invasion. Based on the multivariate model, we were able to develop a prognostic score for seminal vesicle invasion, which allowed us to discriminate two patient groups: A group with low risk of seminal vesicles invasion (5.7%), and the second with a higher risk of seminal vesicles invasion (32.7%). CONCLUSIONS: Using the number of positive biopsies, Gleason score and PSA, it is possible to identify patients with low risk of seminal vesicles invasion. In this population, seminal vesicles might be excluded as a target volume in radiation therapy of prostate cancer.