The impact of centralization of services on treatment delay in ovarian cancer: A study on process quality.

OBJECTIVE: Emphasis on improving healthcare quality has led to centralization of services for patients suspected of ovarian cancer. As centralization of services may induce treatment delays, we aimed to assess compliance with health system interval guidelines in patients suspected of ovarian cancer. DESIGN: Evaluation of health system intervals, comparison between direct and indirect referrals and between 2013 and 2014. SETTING: A managed clinical network (MCN) comprising 11 hospitals in the Netherlands. PARTICIPANTS: Patients that were treated for ovarian cancer within the University Medical Center Groningen in 2013 and 2014. INTERVENTION: Introduction of an MCN to centralize services for patients suspected of ovarian cancer. MAIN OUTCOME MEASURE: Compliance with national guidelines regarding health system intervals. RESULTS: Between 2013 and 2014 a clinically relevant improvement in compliance with guidelines was demonstrated. Within this period, median treatment intervals decreased from 34 to 29 days, and the percentage of patients in which treatment interval guidelines were met increased from 63.5 to 72.2%. New regulations and increased awareness of health system intervals inspired changes in local practice leading to improved compliance with guidelines. Compliance was highest in patients that were directly referred to our academic hospital. CONCLUSION: Evaluation of health system intervals in patients suspected of ovarian cancer was feasible and may be applicable to other MCNs. Though compliance with guidelines improved within the study period, there is potential for improvement. To facilitate real-time evaluation of compliance with national guidelines establishing uniformity of electronic patient files in the MCN is deemed essential.

Definition of unique and shared T-cell defined tumor antigens in human renal cell carcinoma.

From peripheral blood mononuclear cells of a patient with renal cell carcinoma (RCC), we isolated several T-cell clones, which efficiently lyse the autologous RCC cell line (LE-8915-RCC), but not the autologous Epstein Barr virus-transformed lymphoblastoid cell line. Most of the cytotoxic T lymphocyte (CTL) clones recognize HLA-A1-positive allogeneic RCC cell lines, indicating that HLA-A1 is the restricting element for these T cells. One CTL clone exclusively recognizes the autologous tumor cells. The HLA-A1-restricted CTL clones can be divided further into two subsets of T-cell clones, one blocked by an HLA-A1-specific monoclonal antibody, the other not. The reactivity of HLA-A1-restricted T-cell clone 6/135 was studied in greater detail. This T-cell clone also recognizes a number of melanoma cell lines, indicating that expression of the antigen seen by this CTL clone is not restricted to RCC. Strikingly, the antigen is not exclusively expressed by tumor cell lines, because primary cultures of proximal tubulus epithelium cells, adult mesangial cells, and normal breast epithelium cells are also lysed. These results corroborate the notion that renal carcinoma cells are immunogenic by virtue of a broadly distributed antigenic structure that may serve as a target for cytotoxic T cells and may be a potential candidate for tumor vaccine development.

Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.

In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.

Transfection of IL-2 augments CTL response to human melanoma cells in vitro: immunological characterization of a melanoma vaccine.

We have transfected human melanoma cell line 518A2 with the cDNA encoding interleukin-2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF), and compared cytokine-producing clones for their ability to induce melanoma-specific cytotoxic T lymphocytes (CTL) from autologous peripheral blood mononuclear cells (PBMC) in vitro. The parental cell line expressed HLA-A1, HLA-A2, ICAM-1, LFA-3, in addition to the common CTL antigens MAGE-1, MAGE-3, tyrosinase, gp100, and Melan-A/MART-1. Stimulation of autologous PBMC responders with the IL-2-transfected clone 518/IL2.14 specifically induced CTL lines reactive with all cell lines derived from the autologous patient. Strikingly, GM-CSF-transfected 518A2 cells did not induce anti-tumor CTL reactivity. CTL induction against 518/IL2.14 was independent of HLA class II expression or CD4 help. The parental cell line 518A2 gained immunogenic properties when high concentrations of IL-2 were supplied exogenously, indicating that IL-2 produced and present at high levels locally by itself enhanced immunogenicity. From the autologous CTL line reactive with 518/IL2.14, clones were generated against an as yet unknown antigen, which was present in all autologous melanoma cell lines as well as in 7 of 15 HLA-A2+ melanoma cell lines tested, but not in melanocytes. These results will be discussed with respect to the possibility of using IL-2-transfected melanoma cells as a vaccine for treatment of patients with melanoma.

Transcription of the gene encoding melanoma-associated antigen gp100 in tissues and cell lines other than those of the melanocytic lineage.

The expression of the gp100 antigen is generally thought to be confined to cells of the melanocytic lineage, which makes the protein a suitable melanoma-specific marker. Strikingly, after screening a panel of normal tissues, tumour samples and cell lines of non-melanocytic origin, we found transcripts encoding gp100 in virtually every tissue and cell line tested. In contrast, tyrosinase and MART-1/MelanA transcripts were detected only in cells of the melanocytic lineage. However, no gp100 protein could be detected by either Western blotting or cytotoxicity assays. Therefore, at the protein level, gp100 remains exclusive for cells of melanocytic origin despite its transcription in many cell types. The major implication of this finding is that screening of patient material for gp100 expression should preferrably be performed by antibody staining. Reverse transcriptase polymerase chain reaction (RT-PCR) can be employed, provided that it is performed in a tightly controlled, semiquantitative setting.

Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Melanoma and renal-cell carcinoma (RCC) are generally considered to be relatively immunogenic tumor types in humans. In the case of melanoma, many major histocompatibility complex (MHC) class I-restricted tumor-specific cytotoxic T lymphocytes (CTL) have been isolated from either tumor-infiltrating lymphocytes (TIL) or autologous peripheral blood lymphocytes (PBL). In contrast, such CTL have only incidentally been described in the case of RCC. It has often been reported that TIL lines isolated from RCC display non-MHC-restricted and non-specific activity. Here, we report the isolation and characterization of tumor-specific CTL from PBL of one RCC patient and from TIL of another RCC patient. CTL clones 263/17 and 263/45, isolated from the PBL of patient LE-9211, were restricted by HLA-B7. CTL clone 5E, isolated from the TIL of patient LE-8915, was restricted by HLA-B37. The autologous RCC cell lines were efficiently lysed by the CTL clones, whereas normal epithelial cells of the proximal tubuli matched for the restriction element and K562 were not. From a panel of allogeneic RCC cell lines, CTL 5E recognized MZ-1940-RCC. Reactivity to allogeneic RCC sharing HLA-B7 was also observed with CTL 263/17 and 263/45, both of which could lyse the HLA-B7-positive cell line MZ-1851-RCC. Our data provide evidence that common tumor antigens are recognized by CTL on RCC.

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma.

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented by HLA-B7 and is encoded by a new gene that we have named RAGE1. No expression of RAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.

Some association properties of bovine SH-kappa-casein.

(1) It is shown that kappa-casein association is characterized by a critical micelle concentration which decreases as the ionic strength is increased. (2) The kappa-casein polymer molecular weight was calculated from the weight-average apparent molecular weight by taking into consideration the monomer concentration and the excluded volume. The degree of polymerization is 30 and does not depend on ionic strength between 0.1 and 1 M. (3) The non-electrical contribution to the standard free energy of association is -38 kJ/mol monomer. The electrical part is small: 1-2 kJ/mol monomer depending on the ionic strength and kappa-casein genetic variant. (4) The limitation of size and the size itself of the kappa-casein polymer can be explained by the theory of self-assembly of virus particles by Caspar and Klug (D.L.D. Caspar and A. Klug, Cold Spring Harb. Symp. Quant. Biol. 27 (1962)1). (5) Extending this theory to casein micelle assembly, it is predicted that micelles are distributed preferentially over a restricted number of sizes.

Purification and some physicochemical properties of bovine kappa-casein.

1. A description is given of the fractionation of kappa-casein on DEAE-cellulose using a pH gradient. With this method an improved separation of the kappa-casein components with a higher negative charge is obtained. 2. It is shown that at least one of the kappa-casein fractions has a second phosphate ester group. The heterogeneity of kappa-casein therefore is not exclusively caused by a varying N-acetylneuraminic acid content. 3. Ultracentrifuge experiments and exclusion gel chromatography show that the purified kappa-casein fraction having the lowest electrophoretic mobility exhibits a monomer-polymer association equilibrium. The free energy of association per mol monomer in 0.2 M NaCl is approximately --36 kJ-mol-1.