Prognostic classification of malignant melanomas by combining clinical, histological, and immunohistochemical parameters.

In a retrospective study the prognostic relevance of clinical, histopathological, immunohistochemical, and flow-cytometric parameters in primary malignant melanomas was evaluated using both the receiver operating characteristic ROC procedure and the logistic regression model. The proteolytic enzymes collagenase IV, cathepsin B, and cathepsin D proved to be significant prognostic factors. Combining the results obtained with these enzymes with gender, anatomic site, tumour thickness, Clark's level, ulceration, pattern of invasive growth, and presence of large round cells resulted in greatly improved discrimination between metastasized and non-metastasized cases. It is anticipated that this method could allow for precise individual prognostic characterization and in particular for identification of high-risk patients for adjuvant therapy.

Quantification of cathepsin D in plasma of patients with malignant melanoma.

The high mortality rate of melanoma patients who develop metastases prompted us to seek for a prognostic soluble marker to identify high-risk and non-risk patients at the stage of the primary tumour. Therefore, we developed a new ELISA for quantifying plasma concentrations of the proteolytic enzyme cathepsin D (CD) in patients with primary tumours (MM-P) and with metastases (MM-M), respectively, compared to a control group. Whereas healthy probands (n = 56) and MM-P (n = 68) showed similar mean values of CD (0.73 +/- 0.45 ng/ml and 0.82 + 0.80 ng/ml), MM-M (n = 40) yielded significantly reduced plasma levels (0.43 +/- 0.53 ng/ml) revealing a high significant discrimination both between controls and MM-M, and MM-P and MM-M (p < 0.0001). From the beginning of the study (1990) to the present 11 of 68 MM-P developed metastases. In order to test the prognostic efficiency of this enzyme to determine those patients at high-risk and non-risk for developing metastases, the receiver operating characteristic analysis was used showing that CD plasma levels cannot supply reliable prognostic values (W = 0.53, p = 0.66).

Soluble HLA class I antigens in plasma of patients with malignant melanoma.

The high mortality rate of melanoma patients who develop metastases prompted us to look for prognostic markers to determine high-risk and non-risk patients at the primary tumour stage. Therefore, we quantified plasma concentrations of soluble HLA class I antigens (sHLA-I) by ELISA in patients with primary tumours (MM-P) and with metastases (MM-M), respectively, and compared them to a control group. Whereas healthy probands (n = 55) and MM-M (n = 38) showed similar mean values of sHLA-I (1.30 +/- 1.44 micrograms/ml and 1.29 +/- 1.27 micrograms/ml), MM-P (n = 67) revealed significantly reduced levels of this marker (0.84 +/- 0.85 microgram/ml). This result matches with our immunohistological staining of membrane-bound HLA-I in sections of paraffin-embedded melanoma. Further subdivision of the MM-P substantiated the observation that mean values of decreased sHLA-I concentrations are in line with high tumour thickness. Since the beginning of this study (1990) to date, 11 of 67 MM-P have developed metastases. The prognostic efficiency of sHLA-I to identify high-risk and non-risk patients was tested by ROC-analysis (receiver operating characteristic) and did not demonstrate good prognostic relevance for sHLA-I (W = 0.64, p = 0.04).

Soluble HLA class I and class II antigens in patients with multiple sclerosis.

Soluble HLA class I (sHLA-I) and soluble HLA class II (sHLA-II) antigen levels during different stages of disease were investigated in paired serum and cerebrospinal fluid (CSF) samples from 37 patients with multiple sclerosis (MS) using ELISA and Western blot analysis. Soluble HLA-II antigens in the serum of untreated patients with the relapsing-remitting type of MS (RRMS) were found to be significantly elevated in acute relapse as compared to values obtained from patients under steroid treatment, in remission or healthy controls. No significant differences in circulating sHLA-I levels could be detected. In contrast, a trend towards increased intrathecal production of sHLA-I molecules in the CSF was observed in untreated RRMS patients in acute relapse, whereas the levels of soluble HLA-II antigens in the CSF were below the detection limit of the ELISA method. Our observations underline the presence of systemic immune activation in MS patients, as reflected in elevated serum sHLA-II antigen levels, and reveal a dichotomy between sHLA class I and II antigen production in the peripheral blood versus CSF in acute MS. Serial measurements of sHLA-II antigen levels might represent a non-invasive method to assess disease activity in MS patients.

Association of increased bronchoalveolar lavage fluid albumin and serum beta 2-microglobulin with pulmonary complications after allogeneic bone marrow transplantation.

The aim of this prospective study was to identify markers in bronchoalveolar lavage fluid (BAL fluid) and serum predictive for the development of pulmonary complications in the early phase (< 50 days) post-BMT. Concentrations of BAL fluid albumin (alb) and serum beta 2-microglobulin (S-beta 2m,) were determined 10 days before BMT (BAL-B, baseline) and on day 1 post-BMT (BAL-1) in 20 patients who subsequently developed pulmonary complications (group 1) and in 66 patients who remained free of complications for a minimum of 12 months (group 2). Median BAL fluid alb concentrations were significantly (P < 0.05) higher in group 1 patients as compared to group 2 patients at BAL-B (40 vs 28 mg/l) and at BAL-1 (30 vs 15 mg/l). S-beta 2m at BAL-1 was also significantly elevated in group 1 patients (median 1.3 mg/l) compared to group 2 patients (median 1.15 mg/l). Using cut-off values for BAL fluid alb (> 23 mg/l) and S-beta 2m (> 0.8 mg/l) we identified 12 patients out of 19 who developed subsequent pulmonary complications from 12 out of 62 patients without such complications, 1 day post-BMT.

Soluble HLA class I and class II concentrations in factor VIII and PCC preparations.

Soluble HLA class I (sHLA-CI) and class II (sHLA-CII) molecules were quantitated in 11 commercially available factor VIII and prothrombin complex concentrate (PCC) preparations by enzyme-linked immunosorbent assays (ELISA). In 4 preparations, we detected traces of sHLA-CI (0.01-0.07 mg/l), and in 7 hemostatic preparations small amounts of sHLA-CII molecules (0.02-0.28 mg/l). The concentrations of these contaminant molecules are unequivocally below the mean values of sHLA in human plasma (sHLA-CI: 1.01 +/- 0.72 mg/l; sHLA-CII: 1.53 +/- 2.44 mg/l). Based on the total amount of chronically administered factor VIII or PCC, contaminating sHLA molecules may be in principle able to exert immunomodulatory effects in patients treated with such preparations.

Soluble HLA class I and beta-2-microglobulin plasma concentrations during interferon treatment of chronic myelogenous leukemia.

Soluble class I molecules (sHLA-ABC) were measured by an enzyme-linked immunosorbent assay (ELISA) in plasma samples of 13 patients with chronic-phase Ph1-positive chronic myelogenous leukemia (CML). The patients were treated once daily with interferon (IFN) s.c. at a dosage of 4 x 10(6) IU/m2 IFN-alpha-2b or in combination with 50 micrograms IFN-gamma. Measurements were performed before 2, 4, 6, 8, 24, 48, and 72 h after the start of treatment and thereafter every 2-4 weeks. Baseline sHLA-ABC levels were within normal limits (mean 22.1 +/- 8.8 mg/l). An initial decrease of sHLA-ABC (mean 3.2 +/- 2.7 mg/l) was seen in all patients during the first 2-8 h of IFN treatment. Thereafter, sHLA-ABC levels increased steadily reaching maximum values within 2-5 weeks. The overall increase was 12.7 +/- 12.4 mg/l. During the following 2-4 months of IFN treatment sHLA-ABC decreased to near baseline levels in 12 of 13 patients. No difference was detected between IFN-alpha and IFN-alpha plus IFN-gamma treatment. beta 2-Microglobulin values were measured in 8 patients and were found to be correlated to sHLA-ABC concentrations (r = 0.48).

Summary report from the first international workshop on soluble HLA antigens. Paris, August 1992.

The First International Workshop on Soluble HLA antigens focused on the comparison of immunoassay procedures for quantitation of soluble HLA (sHLA) class I antigens and the selection of a sHLA class I antigen international standard. Several sets of serum, plasma, and cell culture supernatant specimens were assayed blindly for levels of sHLA class I antigens by 15 participating laboratories using different immunoassay formats. The sandwich ELISA using (i) for antigen capture: an anti-HLA class I heavy chain monoclonal antibody (mAb) specific for a monomorphic epitope, and (ii) for antigen detection: an anti-beta 2 microglobulin antibody-enzyme conjugate, was the assay format of choice. There was a high inter-laboratory correlation among the majority of laboratories. All serum and plasma specimens from normal donors, and from a single transplant patient, had detectable levels of sHLA class I antigens. Paired serum and plasma specimens had similar levels of sHLA class I antigens, although plasma sHLA antigens seemed more stable than serum sHLA antigens. sHLA-A2 and sHLA-B7 antigens were detected in all specimens from HLA-A2 and HLA-B7 donors, respectively, using allele-specific ELISAs. No difference in reactivity was observed for quantitation of native sHLA class I antigens whether the capture mAb was TP25.99 (alpha 3 domain-specific) or W6/32 (alpha 2 + alpha 3-specific). However, a human-mouse chimeric sHLA class I antigen reacted weakly in assays which used TP25.99 mAb. The wide variation among laboratories in their reporting of micrograms/ml units pointed to the need for an inter-laboratory standardization based on a calibrated sHLA antigen preparation. T.sB7, an sHLA-B7 antigen derived from a cell line transfected within human beta 2 microglobulin and HLA-B7 genes, was accepted as the First sHLA class I Antigen International Standard at the workshop meeting.

Soluble CD4, CD8, and HLA molecules in commercial immunoglobulin preparations.

CD4 and CD8 exist in membrane-bound and soluble forms. These antigens are the physiological ligands for molecules of HLA classes I and II, which we have found in soluble form in commercial immunoglobulin preparations. We measured concentrations of soluble CD4 and CD8, as well as soluble HLA-ABC and HLA-RQP in sixteen immunoglobulin preparations by enzyme immunosorbent assays. Only two preparations contained detectable CD8, and nine had soluble CD4 in varying amounts. There was great variation in concentrations of soluble HLA molecules. It is possible that the immunomodulating effects of immunoglobulin therapy in disorders with immune aetiology are due, at least partly, to these contaminating molecules.

Lack of HLA-molecules on human spermatozoa and in seminal plasma.

The expression of human leukocyte antigens (HLA) on ejaculated spermatozoa and on lymphocytes was compared by flow cytometry using monoclonal antibodies towards HLA class I (pan-HLA-A, -B, -C) and class II (DR) antigens. Soluble antigens of HLA class I (s HLA-A, -B, -C) in seminal plasma and in blood plasma were monitored with an ELISA technique. Lymphocytes showed specific fluorescence after incubation with the antibodies against HLA class I and class II (DR), whereas, on spermatozoa no positive immunofluorescence could be detected. No antibodies were bound to any significant extent either after modifications of sperm preparation (density gradient centrifugation, swim up-technique, addition of azide, foetal calf serum or benzamidine chloride) or after treatment of spermatozoa with detergents. Furthermore, different concentrations of soluble HLA-A, -B, -C in seminal plasma and in blood plasma were detected. The latter one showed soluble HLA about four-fold more concentrated than the seminal plasma (means +/- SD: 262.5 +/- 144.4 nmol l-1 vs. 62.5 +/- 27.1 nmol l-1). These results suggest, that the HLA-expression differs between human spermatozoa and somatic cells.

Soluble HLA class I and class II concentrations in commercial immunoglobulin preparations.

Soluble HLA class I (sHLA-ABC) and class II (sHLA-RQP) molecules were quantitated in 16 commercially available immunoglobulin (Ig) preparations by enzyme-linked immunosorbent assays. Whereas three Ig preparations contained no detectable sHLA-ABC, all preparations showed concomitant sHLA-RQP molecules. There was a considerable variability with regard to the individual sHLA concentrations. For sHLA-RQP the values exceeded that found in human plasma of healthy individuals, suggesting that the extraction procedure may concentrate not only Ig, but also HLA class II molecules. Based on the total dosage of intravenously administered immunoglobulins (i.v.Ig), contaminating sHLA molecules may become immunogenic. Furthermore, sHLA molecules are discussed in terms of participation in the well-known immunomodulating effects of i.v.Ig therapy.

Enhanced serum levels of soluble HLA class I molecules are induced by treatment with recombinant interferon-gamma (IFN-gamma).

In order to investigate serum levels of soluble HLA class I antigens after single injection of various doses of recombinant IFN-gamma (rIFN-gamma) and to correlate the changes observed to beta-2-microglobulin serum levels, we studied five patients with metastasizing renal cell carcinoma. Each patient received three treatment cycles of 10, 100 and 500 micrograms rIFN-gamma three times at weekly intervals. The treatment cycles were separated by a therapy-free interval of 2 weeks. The order of dose levels was randomly assigned to each patient. Serum levels of soluble HLA class I proteins were measured by an ELISA in samples drawn immediately before and 4, 24, 48, 72 and 168 h after each administration of rIFN-gamma. Beta-2-microglobulin was assessed in parallel using a commercially available radioimmunoassay. Significant induction of soluble HLA class I protein serum levels was observed after treatment with 100 and 500 micrograms rIFN-gamma. The increments peaked after 2-4 days and remained elevated for up to more than 7 days. A significant correlation between increments of soluble HLA class I proteins and beta-2-microglobulin was observed. We conclude that measurement of soluble HLA serum levels is practical for monitoring induction of HLA class I synthesis in patients treated with rIFN-gamma. The correlation observed between induction of beta-2-microglobulin and soluble HLA class I proteins indicates that measurement of beta-2-microglobulin might be sufficient for the biological response monitoring in clinical studies.

Quantitation of soluble HLA class II molecules by an enzyme-linked immunosorbent assay.

In order to quantify soluble HLA-DR,DQ,DP molecules (sHLA-RQP) an enzyme-linked immunosorbent assay was developed utilizing two monoclonal antibodies specific for HLA-DR,DP (Tü35) and HLA-DQ (Tü22) gene products, respectively. Highly purified HLA class II molecules isolated from a lymphoblastoid cell line were used for calculation of exact sHLA-RQP protein values. Circadian variations of sHLA-RQP plasma levels were studied in 7 healthy probands showing no significant deviations; measurements in 4 probands at intervals between 4 and 6 weeks revealed that sHLA-RQP levels remain relatively stable. The population analysis of 209 unrelated, HLA-typed healthy donors resulted in an average protein concentration of 1.53 +/- 2.44 micrograms/ml plasma for sHLA-RQP. Four out of 209 probands (= 1.9%) had no detectable sHLA-RQP. Significant associations of high or low sHLA-RQP levels to particular HLA-DR or -DQ specificities were not observed. However, plasma derived from HLA-DR9 positive had the highest and from HLA-DR8 positive donors the lowest mean sHLA-RQP values. By comparing HLA identical with two-haplotype-different siblings we found no evidence that sHLA-RQP plasma levels are under genetic control of the HLA complex or closely linked genes. Furthermore, soluble HLA class I plasma concentrations in 100 probands analyzed showed no correlation to those of sHLA-RQP.

Quantification of soluble HLA class I gene products by an enzyme linked immunosorbent assay.

A simplified enzyme linked immunosorbent assay utilizing an HLA class I framework-specific monoclonal antibody and a polyclonal enzyme linked beta-2 microglobulin specific antiserum has been established for the quantitative measurement of soluble HLA class I molecules. A total of 219 unrelated healthy individuals and 137 members of 28 families typed for HLA were analyzed for their non-membrane bound, i.e. soluble HLA-A,B,C antigens (sHLA-A,B,C). As reported by others, we observed associations of higher or lower sHLA-A,B,C values to particular HLA antigens: High plasma values were observed in probands positive for HLA-A23, A24, A29, Aw33, Bw65, and Cw8 and low values in HLA-B27 and B37 positive individuals. However, as shown by family studies, levels of sHLA-A,B,C were apparently not controlled by the MHC haplotypes alone, since no significant difference between HLA identical siblings and two haplotype different individuals could be detected. Thus, additional non-MHC linked gene(s) may be involved in the release of class I gene products.