Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGß, hCGßn, hCGßcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.

The usefulness of detecting thyroglobulin in fine-needle aspirates from patients with neck lesions using a sensitive thyroglobulin assay.

The aim of this study was to assess the diagnostic utility of thyroglobulin (Tg) in fine needle aspirates (Tg-FNAB) of nonthyroidal neck masses using a sensitive in-house method for detecting Tg in washout specimens. A total of 256 samples from 145 patients were evaluated for Tg in washout specimen from FNAB and compared to corresponding cytological smear and histology of 46 surgical specimens. Tg was measured by a sensitive in-house time-resolved immunofluorometric assay. The sensitivity for Tg-FNAB alone or in combination with cytological findings was found to be 100% in both the follow-up group and before primary surgery. In the follow-up group the specificity of Tg-FNAB was 100%. Fifty-nine of 60 follow-up specimens with malignant cytology were Tg-FNAB positive (n = 195). Histological examination of one lymph node with malignant cytology and negative Tg-FNAB showed metastasis from carcinoma of the salivary gland. Tg-FNAB was positive in 25 specimens with suspicious or cystic cytology. Tg-FNAB values were high (median 4557 microg/l, range 122-37200 microg/l) in washout specimen from cystic metastasis from which cytology did not confirm malignancy. Of the 20 lymph nodes with histology confirming metastasis from differentiated thyroid carcinoma (DTC), the Tg-FNAB was positive in 19 and intermediate in one. However, before primary surgery, two Tg-FNABs were false positive compared to the histology of the lymph nodes. TgAb in serum did not interfere with FNAB-Tg measurements. Tg-FNAB measurement is accurate with high sensitivity (100%) and of great importance in detecting cystic metastasis when cytology is not conclusive. Even metastases to small neck lymph nodes may be detected by using sensitive Tg-assay. Serum thyroglobulin antibodies appear to have ignorable effect on the clinical performance of Tg-FNAB.

Alpha fetoprotein is increasing with age in ataxia-telangiectasia.

The elevated serum alpha fetoprotein (AFP) concentration in ataxia-telangiectasia (A-T) patients has been known for decades, but the individual variation of AFP levels over time has not been studied. We have followed 12 patients (five girls and seven boys) for 1-12 years (mean 5.5 years) measuring in each patient AFP 2-8 (mean 4) times. Serum AFP levels were increased in all patients, mean 168.7 (range 40-373) kU/L, and without significant differences between the patients. There was a significant age related difference in the serum AFP level. A positive linear relationship (r=0.61, p=0.04) could be found between AFP level and age. Albumin levels were within normal range and did not change with age. Four patients had slightly increased aspartate aminotransferase (AST) levels. None of the patients had serological evidence of infectious hepatitis, and none had increased levels of carcinoembryonic antigen. Repeated standardized observations of gait function revealed no major difference in neurological deterioration between our patients. All had classical A-T disease and mainly truncating mutations; 21 out of 24 possible mutations were either frameshift or nonsense. Four were homozygous for the Norwegian ATM founder mutation. No correlation between serum AFP levels and the different ATM genotypes could be found. We conclude that serum AFP is not only elevated, but also is continuously increasing with age in patients with classical A-T disease.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

The ISOBM TD-7 Workshop on hCG and related molecules. Towards user-oriented standardization of pregnancy and tumor diagnosis: assignment of epitopes to the three-dimensional structure of diagnostically and commercially relevant monoclonal antibodies directed against human chorionic gonadotropin and derivatives.

The ISOBM TD-7 hCG Workshop was established to characterize the molecular epitope structure and specificities of a panel of diagnostically relevant monoclonal antibodies (MAbs) directed against human chorionic gonadotropin (hCG) and its derivatives, and to consider how this information could be used to improve comparability of immunoassay results for these analytes. In this multicenter study, 27 MAbs have been characterized in detail as to their main and fine specificities by direct binding-, competitive- and sandwich-RIA, -ELISA, BIAcore and Western blotting. Antigens used in the study included the upcoming first WHO reference reagents for immunoassay, i.e. nick-free hCG (hCG), nicked hCG (hCGn), hCG alpha-subunit (hCGalpha), hCG beta-subunit (hCGbeta), nicked hCG beta-subunit (hCGbetan), hCG beta-core fragment (hCGbetacf), synthetic peptides of hCGbeta C-terminal peptide (hCGbetaCTP), and homologous hormones, luteinizing hormone (LH) and subunits (LHbeta) from various species. Correct classification of blinded internal controls demonstrated the reliability of the MAb referencing approach. Three-dimensional molecular epitope assignment was possible in many instances by comparing immunoreactivity of the ISOBM MAbs (n = 27) to a large panel of MAbs (n = 18) previously well characterized in the Innsbruck (P.B.) and Paris (J.M.B.) laboratories. All three major antibody specificities (alpha, n = 1; beta, n = 21; alphabeta, n = 5) were represented in the TD-7 MAb panel. HCGbeta MAbs could further be subdivided into (i) those recognizing hCGbeta only (epitopes: beta(6), n = 1; beta(7), n = 2; beta(14), n = 1) and (ii) those recognizing hCGbeta + hCG (beta1, beta2, beta4, beta5, n = 10; beta8 and beta9, n = 9). Members of the latter group were specific either for hCG + hCGbeta + hCGbetacf (beta1, n = 3) or hCG + hCGbeta + hCGbetaCTP (beta8, n = 6; beta9, n = 1) or in addition to hCG + hCGbeta + hCGbetacf recognized hLH/hLHbeta to a minor (beta2, n = 3; beta4, n = 3) or similar degree (beta5, n = 1). Epitopes were (i) located on the first and third loops protruding from the cystine knot of hCGbeta (beta2-beta6, aa hCGbeta20-25 and 68-77), (ii) presumably centered around the knot itself (beta1), or (iii) on hCGbetaCTP (epitope beta8 = hCGbeta141-144, beta9 = hCGbeta113-116). The ISOBM panel of MAbs represents all major epitope specificities suitable for the design of specific sandwich immunoassays. High analyte variability in serum and urine during the course of pregnancy and tumor development favors certain epitope combinations. For routine diagnostic purposes, assays recognizing a broad spectrum of hCG/hCGbeta variants such as hCG + hCGn + hCGbeta + hCGbetan + hCGbetacf + -CTPhCG + -CTPhCGbeta may be useful. Low cross-reactivity against related glycoprotein hormones (e.g. hLH) and their derivatives is mandatory. These criteria are best met by combinations of MAbs directed against epitopes located around the cystine knot (beta1) and against those encompassing the top of loops 1 and 3 on hCGbeta (beta2, beta4). The first WHO reference reagents for immunoassay of hCG and hCG-related molecules being prepared by the IFCC should facilitate characterization of what assays for 'hCG' are measuring. The next step towards improving between-laboratory comparability of measurements of hCG/hCG derivatives in pregnancy and oncology is provided by results of this TD-7 Workshop.

Prognostic value of neuroendocrine serum markers and PSA in irradiated patients with pN0 localized prostate cancer.

BACKGROUND: The prognosis of patients with localized prostate cancer depends on clinical stage, histological grade, and pretreatment prostate-specific antigen (PSA). We evaluated the additional prognostic impact of serum levels of neuron-specific enolase (NSE) and chromograninA (CgA) after curative radiotherapy and the importance of serum PSA, analyzed 3 months after irradiation. METHODS: From 1988 to 1995, 161 patients with localized T1-4, pN0M0, prostate adenocarcinoma were treated with external radiation (66Gy, 2Gy/5 fractions per week). Frozen serum samples were assessed for CgA, NSE, and PSA before and 3 months after radiotherapy. CgA was analyzed in only 100 patients. NSE and CgA were determined by a immunometric assay. Total PSA was measured by a time-resolved fluoro-immunometric assay. RESULTS: Prior to radiotherapy CgA was elevated in 16 of 100 patients, and NSE was elevated in 33 of the 161 patients. There was no association between grade, T category or pretreatment PSA and the levels of neuroendocrine markers. Pretreatment-elevated serum NSE, but not initial CgA, identified patients with an unfavorable prognosis. A < 50% reduction of PSA 3 months after radiotherapy was associated with decreased failure-free 10 years urvival. Multivariate analysis demonstrated an increased risk of failure for patients with elevated pretreatment NSE and PSA values, T3 category, and decline of PSA less than 50% 3 months after radiotherapy. The presence of none or several risk factors (1-4) defined clearly separable groups. CONCLUSIONS: Together with T category and pretreatment serum PSA values, serum NSE values before radiotherapy and decrease of serum PSA 3 months after radiotherapy represent easily assessable prognostic parameters in patients undergoing curative radiation treatment for prostate cancer.

Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer.

Four different genes were identified by immunoscreening of a cDNA expression library from the human prostate cancer cell line DU145 with allogeneic sera from four prostate cancer patients. A cDNA encoding the nucleolar protein No55 was further analysed and shown to be expressed at the mRNA level in several normal tissues, including ovaries, pancreas and prostate and in human prostate cancer cell lines PC-3, PC-3m and LNCaP. By reverse transcriptase/polymerase chain reaction, expression of No55 was several-fold higher in two out of nine prostate cancer primary tumours and two out of two metastatic lesions, compared to normal prostate tissue. Antibodies to No55 were detected in sera from seven out of 47 prostate cancer patients but not in sera from 20 healthy male controls. Sequence analysis of the No55 open reading frame from normal and tumour tissues revealed no tumour-specific mutations. The No55 gene was located to chromosome 17q21, a region reported to be partially deleted in prostate cancer. Considering the immunogenicity of the No55 protein in the tumour host, the expression profile and chromosomal localization of the corresponding gene, studies evaluating No55 as a potential antigen for immunological studies in prostate cancer may be warranted.

Cushing's syndrome in prostate cancer. An aggressive course of prostatic malignancy.

We report a case with an initial diagnosis of adenocarcinoma of the prostate in whom Cushing's syndrome developed. The disease did not respond to estrogen treatment and the patient died of severe septicemia. Histopathologic examination of the autopsy specimens revealed a small cell carcinoma intermingled with a moderately differentiated adenocarcinoma in the prostate and widespread metastases of small cell carcinoma. Immunoreactivity for neuroendocrine differentiation was found only in the small cell carcinoma. Determination of different tumor markers in plasma samples showed markedly elevated levels of prostate-specific antigen as well as carcinoembryonic antigen prior to treatment, with no significant changes after treatment. The concentration of the neuroendocrine marker chromogranin A was initially within the normal range, but increased during estrogen treatment, whilst neuron-specific enolase was moderately elevated throughout the observation period.

Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen.

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Reactivity of 77 antibodies to prostate-specific antigen with isoenzymes and complexes of prostate-specific antigen.

Seventy-seven antibodies submitted to the ISOBM TD-3 PSA Workshop (TD-3.1 and TD-3.2) were characterized by measuring their reactivity with isoenzymes of free prostate-specific antigen (PSA), PSA complexed to alpha1-antichymotrypsin (PSA-ACT) and alpha1-proteinase inhibitor (PSA-API). Antibodies were classified into 15 distinct groups according to their reaction profiles with the various isoenzymes. Some antibodies recognizing both free and complexed PSA were inaccurate in measuring total PSA. Eight of the 9 free PSA-specific antibodies cross-reacted more with PSA-API than with PSA-ACT, while 1 antibody reacted less with PSA-API than PSA-ACT. From the panel of antibodies 39 reacted with both free and complexed PSA and were classified as total PSA antibodies.

Epitope mapping and affinity estimation of 83 antibodies against prostate-specific antigen.

Eighty-three antibodies submitted to the ISOBM TD-3 Workshop on prostate-specific antigen (PSA) were characterized by cross-inhibition studies, immunometric assay and affinity estimation with free or complexed PSA (PSA-alpha1-antichymotrypsin, PSA-ACT). Nine antibodies did not bind PSA or PSA-ACT when coated onto microtiter plates or in solution. Another 3 antibodies bound the antigens only when in solution and were therefore omitted from the cross-inhibition experiments. Dissociation constants (Kd) were estimated from the concentration of free antibody needed to achieve half-maximal binding of the antigen. Kd values for PSA and PSA-ACT ranged from 2 x 10(-12) to >10(-8) mol/l. Antibodies were classified into 6 main groups according to their reactivity. Group 1 comprised 15 antibodies (#25, 26, 33, 68, 73, 77, 78, 80, 85, 209, 213, 216, 223, 230, and 262) specific for free PSA. These antibodies had >80% cross-inhibition and showed high affinity for PSA with minimal or no affinity for the PSA-ACT complex. Group 2 comprised antibodies that reacted with both free PSA and PSA-ACT. Three subgroups were defined: group 2a (#40), group 2b (#32) and group 2c (#35, 37, 63, 90, 215 and 226). Group 3a antibodies (#31, 36, 37, 57, 64, 66, 72, 82, 84, 212, 224, 229, 257 and 260) were closely related to those of group 2, with two exceptions in group 3b (#88 and 89). Group 4 contains antibodies with binding patterns similar to those represented by groups 3b and 6b. These antibodies could be divided into two subgroups: group 4a (#30, 38, 51, 217, and 220) and group 4b (#74). Group 5 was more heterogeneous, with distinct inhibition patterns: group 5a (#50, 54, 76, 81,207, and 222); group 5b (#41), and group 5c (#28 and 86). Group 6 antibodies bind epitopes on both free PSA and PSA-ACT, but have epitopes unrelated to those represented in groups 1-3: group 6a contains 15 antibodies (#24, 27, 29, 34, 55, 56, 65, 79, 210, 214, 218, 221, 225, 258 and 261), and group 6b 2 antibodies (#67 and 75). These 6 groups represent the major immunodominant regions, one of which is exposed only on free PSA. Our classification could provide a useful guide in choosing antibodies for future PSA assays.

Specificity and affinity of 30 monoclonal antibodies against alpha-fetoprotein.

Twenty-two antibodies with high affinity for AFP could be classified into groups according to five AFP binding regions, designated A-E, based on cross-inhibition studies and immunometric assay combinations. Antibodies in group A (ISOBM TD2 No. in parentheses): H31 (119), AFP-4-F45 (111), 140/5 (117), K51 (99), K52 (110), AFP-200014A (120) and F2 (118) and in group B: A4-4 (98) and AFP-4-F67 (93) were only inhibited by antibodies belonging to the same groups and could be used in immunometric assay combinations with all other antibodies. Groups C, D and E were inhibited by antibodies in adjacent antibody groups and did not form immunometric assay pairs with antibodies belonging to neighboring groups. Group C comprises: K28 (105), A34-B/B5 (101), AFP-4-F111 (95), AFP100025B (121) and K57 (116), group D: E7 (114) and D10 (92) and group E: H219 (115), K6B1 (103), A34-A/D12 (104), C2 (102), C9 (94) and C10 (97). For six of seven antibodies with low binding to labelled AFP, the specificity could not be determined. Two antibodies were not conclusively assigned to any binding region. Antibody 9A12 (109) may be classified into group E based on immunometric assay combinations, but could not be evaluated in cross-inhibition experiments due to low binding to labelled AFP. Antibody 19F12 (100) functioned as a tracer antibody in combination with all other antibodies, but did not bind AFP when used as solid phase antibody. This antibody could therefore represent a unique binding specificity.

Stability of free and total prostate specific antigen in serum from patients with prostate carcinoma and benign hyperplasia.

PURPOSE: Instability of prostate specific antigen (PSA) in serum might complicate the interpretation of the free-to-total PSA ratio. We studied the in vitro stability of free PSA and total PSA in serum of patients with prostate cancer or benign prostate hyperplasia (BPH), and of elderly men without known prostate disease. Furthermore, we investigated conditions to stabilize the in vitro values in serum. MATERIALS AND METHODS: The effects of storage at 4C on free and total PSA were investigated in serum of 32 men with prostate cancer, 25 with BPH and 29 older than 70 years. All had total PSA less than 25 microg./l. The influence of total PSA levels on in vitro changes in free-to-total PSA was studied in serum of 39 other prostate cancer patients (total PSA 1.7 to 298 microg./l.). Stabilization studies were performed in yet another series of samples from 54 prostate cancer patients (total PSA 1.3 to 238 microg./l.) by adjustment of serum pH to 5.5 before storage. Free and total PSA was measured by a commercial immunofluorometric assay, as well as by in-house immunofluorometric assays. Statistical analyses of the results were performed by analysis of variance with repeated measures. RESULTS: We found no difference between the results obtained by the 2 assay systems. After 7 days at 4C there was a slight decrease in total PSA in sera of prostate cancer patients, BPH patients and men older than 70 years. A decrease in mean free PSA values occurred in all groups (21.3, 15.7 and 14.6%, respectively). The decrease of free PSA with time was significant (p <0.0001) in all groups but there was no significant difference among the groups (p=0.16). The concomitant decrease in free-to-total PSA ratio was significant in all groups (p <0.0001). This change was group dependent (p=0.003), with the largest decrease in the prostate cancer group. Large interindividual differences were observed. Storage at 4C for 7 days of sera of 39 patients with localized and disseminated prostate cancer (total PSA 1.7 to 298 microg./l.) gave a more pronounced decrease in free PSA than in total PSA. Adjustment of serum pH to 5.5 had a stabilizing effect on free PSA and on the free-to-total PSA ratio, giving a significantly smaller change in both values (p <0.0001). CONCLUSIONS: In vitro instability of free PSA in serum and large interindividual differences should be considered when using the ratio of free-to-total PSA in evaluation of patients with suspected prostate cancer. Serum samples should be stored frozen if not analyzed immediately or acidified to pH 5.5. Interpretation of data from determination of free-to-total PSA ratio should be done with caution if the sampling and storage conditions are not known.

Epirubicin combined with estramustine phosphate in hormone-resistant prostate cancer: a phase II study.

Twenty-four assessable patients with hormone-resistant prostate cancer (HRPC) were to receive daily doses of oral estramustine phosphate (EMP), 10 mg kg(-1), and intravenous epirubicin (EPR) infusions, 100 mg m(-2), every third week up to a cumulative dose of 500 mg m(-2). Biochemical response [> or = 50% reduction in pretreatment serum prostate-specific antigen (PSA) after three cycles of > or = 3 weeks' duration] was demonstrated in 13 of 24 patients included (54%). No objective response (WHO criteria) was observed, although seven of nine evaluable patients achieved a > or = 50% serum PSA reduction. Subjective improvement (pain score, performance status) occurred in 7 of 24 patients, whereas nine patients progressed subjectively. There was no correlation between subjective and biochemical response. Biochemical progression (> or = 50% increase of nadir PSA) occurred after a median of 12 weeks. All but two patients were alive after a median follow-up time of 8.7 months for surviving patients (range 3.3-13.2). Eight patients experienced grade 3/4 leucopenia, with no indication of cumulative myelosuppression. Cardiovascular toxicity was experienced by four patients. Two patients developed angioedema twice, in one patient requiring hospitalization at the intensive ward. Based on this limited series, the combination of EPR and EMP in patients with HRPC is tolerable and appears to be effective in terms of significant PSA reduction. The results warrant further investigations of the two drugs and, in particular, of the clinical significance of > or = 50% PSA decrease in patients with HRPC.

Immunohistochemical characterization of 22 monoclonal antibodies against the CA125 antigen: 2nd report from the ISOBM TD-1 Workshop.

We evaluated the immunohistological (IH) characteristics of 22 different antibodies that were submitted for study in the frame of the TD-1 ISOBM Workshop on monoclonal antibodies against CA125. Information on relative affinities and epitope similarities was obtained from a parallel immunochemical study. Antibodies were tested at concentrations of 10 and 1 micrograms/ml on frozen and paraffin sections. Paraffin sections were stained according to the streptavidin-biotin complex protocol, and frozen sections according to a two-step immunoperoxidase technique. Aminoethylcarbazole served as the chromogen. The tissues were from normal proliferative endometrium (formalin-fixed paraffin-embedded) material and clear-cell adenocarcinoma of the ovary (formalin-fixed paraffin-embedded and frozen material). Sections were scored for staining in epithelial cells, basal, apical and diffuse cytoplasmic and in stromal components. Intensity was graded as 1, 2 or 3 for epithelial cells and as -1, -2 or -3 for stroma. The cumulative scores for each antibody expressed the discriminative properties of specific epithelial staining against background. M11 and M11-like antibodies, as well as OC125 and OC125-like antibodies, in general showed good staining results. Although there was a trend for high-affinity antibodies to show higher scores, there was no clear relationship between affinity and staining result. For nine antibodies (ZR45, MA602-1, K102, K94, K90, OV185, K97, K96, OV198), the reactions in paraffin and frozen sections were of similar intensity. Most of these were of low affinity with one exception: antibody ZR45, a rat monoclonal antibody (MAb) which had a high relative affinity. For eight antibodies (M11, K101, MA602-6, ZS33, B27.1, B43.13, K93, OC125), a loss of specific staining was observed in frozen sections. All but two of these antibodies (MA602-6 and OC125) were of high relative affinity. With four antibodies (K91, ZR38, K95, K100), the reverse situation was observed. One (K100) was of low affinity, two (K95 and K91) of high affinity and the fourth (ZR38) was a rat MAb of high affinity. Mainly due to the increased cytoplasmic staining in carcinoma, the reactivity in paraffin sections was less extensive in normal endometrium compared to ovarian carcinoma for the majority of antibodies, irrespective of their affinity or epitope group. The IH characterization of these antibodies may be of help in selecting antibodies with specific properties for further comparative studies. The reactivity of normal endometrium with all useful antibodies makes it a good candidate for standard external IH tissue control.

Specificity and affinity of 26 monoclonal antibodies against the CA 125 antigen: first report from the ISOBM TD-1 workshop. International Society for Oncodevelopmental Biology and Medicine.

The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines. Immunometric assays using all possible antibody combinations were used to study the topography of antibody binding sites on the antigen. We conclude that the CA 125 antigen carries only two major antigenic domains, which classifies the antibodies as OC125-like (group A) or M11-like (group B). One antibody, OV 197, showed binding specificity related to some of the OC125-like antibodies, but was classified into a separate group C. The OC125-like group of antibodies has four subgroups with different binding specificities. These are A1 = OC 125 and K 95, A2 = K 93, A3 = B43.13, and A4 = ZS 33, B27.1 and CCD 247. Binding of nonlabelled OC 125 or K 95 to CA 125 caused a marked increase in binding of labelled OV 197 to the complex. This conformational change was not observed with any other antibody combinations. Antibody B43.13 could form immunometric assay combinations particularly with antibodies of subgroup A4, indicating that the B43.13 epitope is in the periphery of the binding area of OC125-like antibodies. The M11-like group of antibodies is more homogenous with strong cross-inhibition between most antibodies. Only one antibody, ZR 38, would form an immunoassay combination with other M11-like antibodies and thus represents a distinct subgroup. The main group of M11-like antibodies are M 11, ZR 45, MA602-6, K 91, OV 185, K 101, K 90, K 96, K 97, K 102, CCD 242, 145-9, and 130-22. Antibody OV 197 binds to a domain designated C and is unique, as stated above. Antibody pairs from any two of the three groups may be used in immunometric assays. Three antibodies were not studied by complete cross-inhibition due to low affinity (OV 198 and K 100) or lack of material (MA602-1). OV 198 and K 100 are most likely OC125-like and MA602-1 is M11-like. Antibody affinity was estimated with labelled antigen in solution or with antigen absorbed on microtiter wells. Western blot analysis showed staining both in the stacking gel and corresponding to a molecule of 200 kDa. There was a marked difference between the antibodies in their ability to bind to CA 125 immobilized on a membrane. Strongest binding was observed with the M11-like antibodies, particularly M 11, K 96, K 97, MA602-6, 145-9. Antibodies belonging to the subgroup A4 were the only OC 125-like antibodies which reacted well with CA 125 in Western analysis. Digestion of CA 125 with proteolytic enzymes showed it to be particularly sensitive to trypsin cleavage. However, no low molecular weight fragments with preserved immunoreactivity were found.

Expression and interconversion of neuron-specific enolase in patient sera and extracts from small-cell lung cancer cells.

The isoforms of gamma-enolase were characterized in serum from patients with small-cell lung cancer (SCLC) and in extracts from SCLC cell lines and malignant melanoma tumor tissue. Large variations in the expression of the 3 gamma-isoforms of enolase were observed. These forms probably represent the homodimeric gamma gamma-enolase, the heterodimeric alpha gamma-enolase and the monomeric forms of gamma-enolase. Only the dimeric forms are enzymatically active. The predominant gamma-enolase in the cell lines is the heterodimeric alpha gamma-enolase. The SCLC cell lines can be divided into two groups: one with negligible gamma gamma-enolase expression and considerable amounts of the nonneuronal alpha alpha-enolase and a second group with a large fraction of gamma gamma-enolase concomitant with a low expression of alpha-enolase. Similar patterns are observed in tissue extracts from malignant melanoma. When changing buffer conditions by increasing the ionic strength and decreasing the Mg2+ concentration, interconversions between the isozymes occur. In contrast to the predominant alpha gamma-enolase in extracts from cell lines, the multiple forms of gamma-enolase in serum might be caused by a subunit exchange facilitated by the low Mg2+ concentration in plasma. However, there seems to be a stable equilibrium between the isoforms in undiluted patient serum. The induction of subunit exchange by perturbation in ionic strength and/or Mg2+ concentration indicates a need for caution when choosing diluents for use in assays for neuron-specific enolase.

Increase of serum prostatic specific antigen and clinical progression in pN + MO prostate cancer.

OBJECTIVE: To analyse the increase of serum prostatic specific antigen (PSA) as a means of early detection of progression in hormonally untreated or androgen-deprived patients with T1-T4, pN+ and MO prostate cancer. PATIENTS AND METHODS: From 1986 to 1992 40 patients with T1-T4 pN+ MO prostate cancer were either deprived of androgen at diagnosis (Group 1, 19 patients) or had no immediate hormone manipulation (Group 2, 21 patients) and were followed at 3-6-monthly intervals when determinations of PSA and routine clinical/radiological examinations were performed. A significant increase in PSA was defined as a > or = 50% increase of the baseline PSA value which was the either the lowest PSA value within 6 months from the start of androgen deprivation (Group 1) or the initial PSA value (Group 2). RESULTS: By June 1993 22 of the 40 patients had clinically progressed. In 12 patients the progression was preceded by a significant increase in PSA (Group 1, three of four progressing patients; Group 2, nine of 18 progressing patients). A PSA increase of > or = 50% was observed simultaneously with clinical progression in six patients, whereas clinical progression occurred in four patients with no previous or simultaneous significant increase in PSA. In four of nine hormonally untreated patients > or = 1 year elapsed between antecedent PSA increase and clinical progression. CONCLUSION: In routine clinical practice PSA does not significantly increase (> or = 50% of baseline value and > 10 micrograms/L) before disease progression in about one third of patients with pN+ MO prostate cancer managed with or with no hormone manipulation. Future studies should be carried out to determine whether a lower rate of increase in PSA during follow-up has any clinical significance.

Detection of c-erbB-2 related protein in sera from breast cancer patients. Relationship to ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour.

The level of a c-erbB-2 related protein was determined in sera from 168 breast carcinoma patients, 12 females with benign breast disease, and 66 female controls using an ELISA (enzyme linked immunosorbent assay) kit. Elevated c-erbB-2 related protein level was detected in one of 13 preoperative sera (8%), two of 62 postoperative sera from patients without recurrent disease (3%), and 55 of 93 sera collected at recurrent disease (59%). Elevated serum levels were detected significantly more often in patients with distant metastases than in patients with recurrent disease restricted to loco-regional areas (68% versus 19%). Presence of elevated serum level was associated with ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour. None of the patients who had normal ERBB2 gene copy number in tumour had elevated serum levels. Although the usefulness in postoperative prediction of the presence of micrometastases is somewhat questionable, the results suggest c-erbB-2 related protein to represent a novel tumour marker in serum and other body fluids from breast cancer patients at the time of diagnosis and during treatment monitoring.