Ole P. Børmer

Evaluation of serum CA 125 levels in the monitoring of ovarian cancer.

Serum CA 125 levels were evaluated in 227 patients with ovarian cancer. CA 125 levels were elevated in 86% of the patients. All histologic types, including mucinous tumors, were associated with raised CA 125 levels. There was a positive correlation with tumor burden and an inverse correlation with degree of differentiation. In patients undergoing radical operation an elevated CA 125 level was a bad prognostic index. Serial CA 125 measurements were assessable in 112 patients undergoing chemotherapy. Rising or failing levels correlated with disease in 92% of the cases. The CA 125 level increased before clinical progression with a median lead time of 3 months. Only patients who showed objective response to chemotherapy had a decrease in antigen levels of ⩾30% 4 weeks after the first course of chemotherapy and a normalization of CA 125 levels 3 months after initiation of chemotherapy. Rising levels were always associated with progression. These data suggest that CA 125 may aid in early identification of nonresponders. However, a normal CA 125 level does not exclude the presence of disease.

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.

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

Unexpectedly High Serum Methotrexate Levels in Cystectomized Bladder Cancer Patients with an Ileal Conduit Treated with Intermediate Doses of the Drug

The pharmacokinetics of serum methotrexate were studied in 45 bladder cancer patients receiving 250 mg. per m.2 as part of the initial cycle of combination chemotherapy. Serum methotrexate was determined routinely 43 to 49 hours after administration. If the methotrexate levels remained at more than 80 nmol. per l. measurements were repeated daily until the serum levels decreased below this point. The patients were classified into group 1-23 with a bladder in situ and no ureteral obstruction, group 2-11 with a bladder in situ and unilateral hydronephrosis, and group 3-11 who had had cystectomy and ileal conduit diversion before chemotherapy. Of the patients in groups 1 and 2, 5 and 6, respectively, had serum methotrexate levels of 80 nmol. per l. or more 43 to 49 hours after administration, which decreased to below this level on the next day. Of the 11 patients in group 3, 8 had elevated methotrexate levels at the initial determination. Daily methotrexate analyses showed a delayed elimination in 4 of 7 patients and levels of more than 80 nmol. per l. for 3 to 9 days. Low creatinine clearance but, in particular, the previous performance of an ileal conduit predicted high methotrexate levels on day 2 after treatment. The most likely explanation for this observation is the resorption of methotrexate by the small bowel mucosa in the ileal conduit. Patients with an ileal conduit performed 2 years or less before chemotherapy and/or those with a long ileal segment seem to have a particularly high risk for delayed methotrexate elimination. Bladder cancer patients with an ileal conduit who receive methotrexate-containing chemotherapy have a high risk of delayed methotrexate elimination and increased clinical methotrexate toxicity. Leukovorin rescue should be used liberally in these patients together with other prophylactic means (intensive hydration and alkalization of the urine).

Protein Epitopes in Carcinoembryonic Antigen

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.

Reference intervals for carcinoembryonic antigen (CEA), CA125, MUC1, Alfa‐foeto‐protein (AFP), neuron‐specific enolase (NSE) and CA19.9 from the NORIP study

Objective. Adhering to current IFCC recommendations, we calculated upper 97.5 % reference limits for serum tumor markers. Material and methods. Serum samples from 498 healthy individuals from the Nordic reference interval project (NORIP) were investigated for carcinoembryonic antigen (CEA), CA125 and MUC1 (episialin, CA15.3) using in‐house immunofluorometric assays and, for α‐foetoprotein (AFP), a PerkinElmer Life Sciences assay, neuron‐specific enolase (NSE) using an in‐house immunoradiometric assay and CA19.9 using a Beckman Access assay. All assays participate in external quality assessment programs. Results. CEA concentrations increased with age and smoking. Upper reference limits for non‐smokers were 3.59 µg/L at 50 years and 4.12 µg/L at 70 years. CA125 concentrations were age‐independent and the upper reference limit was 35.8 kU/L. MUC1 increased with age and body mass index (BMI). Upper reference limits were 31.7 kU/L at 40 years and BMI 24, 37.5 kU/L at 70 years and BMI 24, and 33.7 kU/L at 40 years and BMI 30. AFP increases with age, and the upper reference limits were 3.82 kU/L at 20 years and 8.70 kU/L at 60 years. An upper reference limit for NSE was 8.91 µg/L in non‐smokers; smokers exhibited significantly lower levels. The upper reference limit for individuals expressing CA19.9 was 28.3 kU/L. Conclusions. For AFP, CA125 and CA19.9, the reference levels obtained were close to previously reported reference ranges. Smoking and age were confirmed as covariates for CEA. The associations between MUC1 with age and BMI and between NSE and smoking have not been reported previously.

Serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes in patients with testicular germ cell tumors

SummaryThe activities of serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes were determined in 252 patients with a history of testicular germ cell tumors (TGCT). Fifteen of 37 patients with TGCT lesions and seven of 215 without had raised levels of S-LDH (above 8.0 μkat/l (480U/l)). Of the patients with TGCT lesions, four had only raised S-LDH-1 levels, one only raise S-LDH-2 (and normal S-LDH), two only raised S-LDH-3 (one with normal S-LDH), and 10 had five combinations of raised levels of S-LDH isoenzymes with a predominance of S-LDH-1. S-LDH and S-LDH-1 correlated significantly with the total tumor volume in the patients with TGCT lesions, especially pronounced in those with lesions from seminoma. Of 34 patients with TGCT metastases, 13 with raised S-LDH levels lived significantly shorter lengths of time than 21 with normal S-LDH. Similarly, 11 with raised S-LDH-1 (above 3.0 μkat/l (180 U/l)) lived significantly shorter times than 23 with normal S-LDH-1. S-LDH is a valuable tumor marker in patients with TGCT, especially in those with seminoma. Routine determination of S-LDH isoenzymes in addition to S-LDH in patients with TGCT is not recommended. In patients with a history of TGCT and an unexplained elevation of S-LDH levels, a raised S-LDH-1 level indicates the presence of TGCT lesions.

CA125 in peritoneal fluid of ovarian cancer patients

The presence of CA125 was assessed in peritoneal fluid from 70 patients with ovarian cancer and 32 control patients. The follow-up period ranged from 39 to 89 months (median, 56 months). The cutoff for normal peritoneal fluid CA125 levels was determined to be 250 U/ml. A positive correlation between the serum and peritoneal fluid CA125 levels was observed (P less than 0.001). Peritoneal fluid levels were higher than serum levels in all patients. Patients with evidence of active ovarian cancer showed higher peritoneal fluid CA125 levels than the control patients (P less than 0.001). Peritoneal fluid CA125 levels correlated inversely with survival (P = 0.004). The peritoneal fluid CA125 levels were higher in patients with bulky tumor than in those with small (less than 1 cm) tumors (P less than 0.001). Eight out of twenty-six patients with active cancer and available peritoneal cytology had a negative peritoneal cytology. Three of these patients showed elevated peritoneal fluid levels. Three patients out of twenty-four showed elevated peritoneal fluid CA125 levels at second-look laparotomy. These 3 patients had negative biopsies at second-look surgery, but relapsed during the observation period. At second-look laparotomy an elevated peritoneal fluid CA125 level may imply a bad prognosis, but a normal level does not exclude the presence of disease.

CA 125 measured before second-look laparotomy is an independent prognostic factor for survival in patients with epithelial ovarian cancer

The prognostic significance of serum CA 125 level measured in the week before second-look operation was evaluated in 208 patients with invasive epithelial ovarian cancer. Serum CA 125 level was greater than 35 U/ml in 44.7% of patients. All patients with pathological complete response (PCR) had a serum CA 125 level less than or equal to 35 U/ml except one who developed lung metastases 2 months later. The sensitivity of serum CA 125 for identifying residual tumor at second-look operations was 58%, the specificity was 98%, the predictive value of a positive test was 99%, and the predictive value of a negative test was 43%. By Cox regression analysis, tumor state of second look, serum CA 125 level, histologic type, FIGO stage, and tumor grade were identified as independent prognostic factors for survival. We conclude that measurement of serum CA 125 level after induction chemotherapy represents a noninvasive method to identify patients at high risk for subsequent death from ovarian cancer. As far as we know, this is the first report to identify serum CA 125 level as an independent prognostic factor at the time of second-look laparotomy.

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-α1-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 × 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.