Protein epitopes in carcinoembryonic antigen. Report of the ISOBM TD8 workshop.

To characterize antigenic sites in carcinoembryonic antigen (CEA) further and to investigate whether there are differences between colon tumor CEA and meconium CEA (NCA-2) that can be detected by anti-CEA monoclonal antibodies (MAb), 19 new anti-CEA MAb were analyzed with respect to specificity, epitope reactivity and affinity. Their reactivities were compared with 10 anti-CEA MAb with known CEA-domain binding specificity that have previously been classified into five nonoverlapping epitope groups, GOLD 1-5. Cross-inhibition assays with antigen-coated microtiter plates and immunoradiometric assays were performed in almost all combinations of MAbs, using conventionally purified CEA (domain structure: N-A1B1-A2B2-A3B3-C) from liver metastasis of colorectal carcinomas, recombinant CEA, meconium CEA (NCA-2), truncated forms of CEA and NCA (CEACAM6) as the antigens. The affinity of the MAbs for CEA was also determined. The new MAbs were generally of high affinity and suitable for immunoassays. Three new MAbs were assigned to GOLD epitope group 5 (N-domain binding), 3 MAbs to group 4 (A1B1 domain), 1 to group 3 (A3B3 domain), 3 to group 2 (A2B2 domain) and 3 to group 1 (also the A3B3 domain). Three MAbs formed a separate group related to group 4, they were classified as GOLD 4' (A1B1 domain binding). The remaining 3 MAbs appear to represent new subspecificities with some relationship to GOLD groups 1, 2 or 4, respectively. Five MAbs, all belonging to epitope group 1 and 3, reacted strongly with tumor CEA but only weakly or not at all with meconium CEA, demonstrating that the two products of the CEA gene differ from each other, probably due to different posttranslational modifications.

Long-term renal function after treatment for malignant germ-cell tumours.

OBJECTIVE: To evaluate prospectively renal function in patients with malignant germ-cell tumours (MGCTs) >10 years after retroperitoneal lymph node dissection alone (RPLND), radiotherapy alone (RAD) or different schedules of cisplatin-based chemotherapy with or without surgery/radiotherapy (CHEM). PATIENTS AND METHODS: In 85 patients, three groups were identified: RPLND, 14; RAD, 18; CHEM, 53, with subdivision of the latter group according to the cumulative cisplatin dose or the additional use of radiotherapy. Renal function was determined by 131Iodine Hippuran clearance or 99m DTPA glomerular filtration rate, and was assessed before treatment and four times during 14 years of follow-up. A value of <70% of the upper limit of the normal range identified impaired renal function. RESULTS: Twenty-five patients displayed long-term impaired renal function, 23 of them from the RAD or CHEM group. In the RAD group, renal function decreased by 8%, whereas a 14% reduction of renal function was observed in the CHEM group. In the CHEM group the cumulative dose of cisplatin, and in the RAD group the age at treatment, were associated with impairment of renal function. Combining all patients, age at treatment and the type of treatment were associated with impaired renal function. CONCLUSIONS: In 20-30% of the patients with germ-cell tumour, standard radiotherapy and chemotherapy strategies are followed by long-term subclinical impaired renal function. These findings support current intentions to avoid overtreatment with these treatment modalities.

[Prophylactic thyroidectomy in carriers of RET oncogene mutation carriers]. / Profylaktisk tyreoidektomi hos baerere av mutasjoner i RET-onkogenet.

BACKGROUND: Medullary thyroid cancer may be inherited dominantly. Germline mutations in the RET oncogene which code for a receptor tyrosine kinase cause MEN2. Thyroidectomy is recommended in family members who carry a mutation. MATERIAL AND METHODS: We have thyroidectomized four children from three families, 12, 10, 7 and 6 years old, because of germline mutations. RESULTS: The 12-year-old had developed a minimal medullary cancer with microscopic lymph node metastases; the others showed variable degrees of C-cell hyperplasia. The mutations were located on exon 10 (C620F) in the two patients from one family, on exon 11 (C634R) in the second family and on exon 14 (V804M) in the third family. In the families with the codon 620 and codon 634 mutations, only medullary thyroid cancer has been diagnosed. In the family with the codon 804 mutation, the index patient has been operated for a pheochromocytoma. The longterm clinical course seems more favorable in the family with the codon 620 mutation than in the two other families. With knowledge of the family mutations, we found that two out of nine family members we previously have thyroidectomized following calcitonin testing did not carry the family mutation. INTERPRETATION: Genetic diagnostic is a safe and reliable predictive test for familial medullary thyroid cancer and should be carried out in any individual with this cancer. Thyroidectomy is recommended in gene carriers at the age of six.

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.

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.

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.

Fecal calprotectin concentration in patients with colorectal carcinoma.

PURPOSE: The study contained herein was undertaken to investigate fecal calprotectin excretion in a series of patients with colorectal carcinoma and to determine whether the excretion was influenced by localization or stage of the tumor. Furthermore, the effect of surgical treatment on the concentrations was studied. Fecal calprotectin was also compared with plasma concentrations of calprotectin, carcinoembryonic antigen, and C-reactive protein. METHODS: Fecal calprotectin was measured in 119 consecutive patients admitted for treatment of colorectal carcinoma. In 116 (97.5 percent) patients, resectional surgery was performed. Plasma calprotectin was measured in 90 (76 percent) patients, carcinoembryonic antigen in 88 (74 percent) patients, and C-reactive protein in 82 (69 percent) patients. RESULTS: Median fecal calprotectin concentration in the 119 patients was 50 (range, 2-950) mg/l, which was significantly (P < 0.0001) higher than in 125 control patients (median, 5.2 mg/l). In 23 patients studied also after resection, the excretion fell greatly. There were no significant differences in fecal calprotectin concentration among patients with different tumor stages. Elevated plasma calprotectin concentrations were found in 67 of 90 (73.3 percent) patients with colorectal carcinoma, compared with elevated fecal calprotectin in 111 of 119 (93.3 percent) patients, and there was no significant correlation between plasma and fecal calprotectin concentrations. Plasma calprotectin concentrations were significantly lower in patients with T1 or T2 tumors than in those with more advanced stages (P = 0.0025). CONCLUSION: Measurement of fecal calprotectin may become a diagnostic tool in detecting colorectal carcinoma. The specificity in relation to colorectal carcinoma has not, however, been completely investigated. Both neoplastic and inflammatory conditions may be associated with elevated values; therefore, it is unlikely that calprotectin can predict specific colonic disorders.

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.

[Tumor marker analyses--valuable tools if used correctly]. / Tumormarkøranalyser--nyttig verktøy ved riktig bruk.

The tumour marker assays in routine use today are not cancer-specific. Most markers are normal products of epithelial tissues, usually excreted from the intact organ. The infiltrating malignant growth allows release directly into lymph and blood, where the product is measured as a "tumour marker". Many tumour markers are glycoproteins depending on the liver function for their catabolism. These mechanisms explain the slight to moderate increases in tumour marker values found in various non-malignant diseases. Therefore, in general, such assays are of no use for screening healthy populations, and a normal value does not exclude cancer. Their main use is in the primary staging of patients known to have cancer, to evaluate the completeness of primary surgery, to follow-up patients for early detection of relapses, and to monitor the effect of cytotoxic or radiation therapy of advanced disease. In all these applications, good clinical judgement is the necessary basis for using these assays.

Evaluation of serum CA 125 level as a tumor marker in borderline tumors of the ovary.

Serum CA 125 was evaluated as a tumor marker in 85 patients with borderline ovarian tumors. Serum CA 125 levels were elevated preoperatively in 18 of 20 (90%) samples (median 66, range 5-272 U ml-1). Preoperative serum CA 125 levels did not correlate to FIGO stage. Preoperative serum CA 125 levels were elevated in seven of nine (78%) with serous tumors (median 131, range 5-272 U ml-1) and in all 11 with mucinous tumors (median 62, range 41-157 U ml-1). There was no significant difference in the CA 125 levels between these two histologic types. Postoperative serum CA 125 levels, measured 3-6 weeks after primary laparotomy, were significantly lower than the preoperative ones (P < 0.001). No difference in the postoperative CA 125 levels was found between those with and those without residual disease after surgery. Postoperative serum CA 125 levels were elevated in eight of 60 (13%) without residual tumor. None of these had relapsed at the time of analysis (26-87 months after surgery). Serum CA 125 levels tended to correlate with disease evolution during chemotherapy. Two with disease remissions had falling levels, one with stable disease had falling level and one with disease progression had rising level. Serum CA 125 samples were obtained before second-look laparotomy in seven patients. Two with negative findings at second-look had normal levels. Of five with positive findings at laparotomy only two had elevated serum CA 125 levels. Disease relapse was associated with elevated serum CA 125 levels in only one of six patients.

Is serum CA 125 at the time of relapse a prognostic indicator for further survival prognosis in patients with ovarian cancer?

The prognostic significance of serum CA 125 level before treatment of relapse for invasive epithelial ovarian cancer has been evaluated in 135 patients. At time of relapse, serum CA 125 level was higher than 35 U/ml in 110 (82%) patients. Those with serum CA 125 level of 35 U/ml or less at relapse had a better prognosis than those with higher values. Among patients with serum CA 125 level higher than 35 U/ml no difference in survival was observed. By multivariate analysis the independent prognostic factors for survival were histologic type (P < 0.0001) and serum CA 125 level (P < 0.01). Randomized trials are needed to further evaluate whether early detection of disease relapse because of serial serum CA 125 measurements does have prognostic significance.

Serum CA 125 level allows early identification of nonresponders during induction chemotherapy.

CA 125 was measured during induction chemotherapy in 119 patients with advanced epithelial ovarian cancer who had residual disease after primary surgery in order to determine whether patients with poor response to further treatment could be identified during early chemotherapy. All patients had a prechemotherapy serum CA 125 level higher than 35 U/ml. Blood samples were further obtained 4 weeks after the first, second, and third course. Four weeks after the second course of chemotherapy, all 20 patients with PCR, or microscopic disease at second-look, all 17 who achieved complete clinical remission, and 36 of 40 who achieved partial remission had serum CA 125 of 65 U/ml or less or had a decrease of 50% or more of the prechemotherapy level. Survival analysis showed that patients with a serum CA 125 level of 65 U/ml or less 4 weeks after the second course of chemotherapy had the best prognosis. In patients with a serum CA 125 level higher 65 U/ml at that time, a decrease of 50% or more of the prechemotherapy level indicated a prognosis better than that with a lesser decrease. The combined criteria for serum CA 125 (level > 65 U/ml 4 weeks after the second course and a decrease < 50% of the prechemotherapy level) allowed for identification of a real high-risk group with a median survival of 8.9 months and was identified by Cox regression multivariate analysis as the most powerful indicator for survival (P < 0.0001).

Serum CA 125 as a tumor marker and the expression of c-erbB-2 oncogene in tubal malignancies.

Serum CA 125 levels were evaluated in 26 patients with fallopian tube malignancies. CA 125 was elevated preoperatively in seven samples (median 178 U ml-1 range 41-19021 U ml-1), and postoperatively in eight of nine (89%) samples collected from patients with residual disease (median 109 U ml-1 range 10-1883 U ml-1) but only in one of seven (14%) samples from patients without residual disease (median 14, range 5-170 U ml-1) (P < 0.001). Changes in the serum CA 125 level during chemotherapy correlated with the clinical course of disease in 13 of 14 patients with a pre-chemotherapy serum CA 125 level> 35 U ml-1. Nine patients with clinical remissions showed decreasing serum CA 125 levels, one with clinically stable disease showed decreasing levels and four with disease progression showed increasing levels. Serum CA 125 levels were measured in four patients before second-look laparotomy. Two of three with positive findings at laparotomy had elevated serum CA 125 levels whilst the third had a normal level. One patient with negative findings at second-look surgery had a normal CA 125 level. Disease relapse was associated with elevated serum CA 125 levels in nine of 10 patients (median 108 U ml-1, range 27-38200 U ml-1). Using immunohistochemical staining, none of the tumors showed positive cytoplasmic staining for c-erbB-2 (NEU) oncogene. This report shows that CA 125 is a reliable tumor marker for monitoring patients with cancer of the fallopian tube during active treatment and follow-up.