[Clinical reasoning and decision-making in practice. A patient with fever and pancytopenia]. / Klinisch denken en beslissen in de praktijk. Een patiënt met koorts en pancytopenie.

An 82-year-old man was admitted with a 1-week history of chilling fever and dry cough. Laboratory tests revealed pancytopenia and elevated levels of C-reactive protein and lactic dehydrogenase (LDH). Screening for infectious diseases was negative. A bone marrow biopsy showed aspecific findings. The combination of pancytopenia, persistent fever, elevated LDH and hepatomegaly (demonstrated by ultrasound examination of the abdomen) was suggestive of the haemophagocytic syndrome. This was confirmed by very high levels of ferritin and soluble interleukin-2 receptor in the blood. In addition, re-examination of the bone marrow showed several haemophagocytic histiocytes. A polymerase chain reaction for Epstein-Barr virus (EBV) revealed a very high viral load. Since the patient had a history of an increased level of anti-EBV immunoglobulin-G, this was explained by a reactivation of the EBV infection. On the sixth day in hospital the patient developed signs of bilateral pneumonia and subsequent multiple organ failure. Despite intensive treatment the patient died. Autopsy revealed no haematological or other malignancies, but did show haemophagocytosis in many organs. It was then concluded that the patient had a virus-associated haemophagocytic syndrome, due to a reactivation of EBV, for which no underlying cause was found.

Cerebral involvement in two patients with Wegener's granulomatosis.

Two cases of cerebral involvement in Wegener's granulomatosis (WG) are described. The course of the disease in both patients was characterized by sudden onset and fatal outcome, despite maximum immunosuppressive therapy. Cerebral involvement is a rare complication of WG. Over the past two decades, only a small number of case-reports appeared of patients with WG who showed this complication. Since the era of cyclophosphamide therapy, it is commonly assumed that cerebral involvement in WG has no influence on patient survival. However, the two patients described here both died shortly after the occurrence of central neurological symptoms.

The influence of combination chemotherapy on antigen expression in ovarian cancer.

The best treatment results for advanced-stage ovarian cancer patients are obtained with cytoreductive surgery followed by combination chemotherapy. However, the 5-year survival rate of only 20% clearly shows a need for new treatment modalities. At present several modes of immunotherapy for cancer are being explored such as the use of monoclonal antibodies, bispecific antibodies, or specific cytotoxic effector cells, all making use of the presence of tumor-associated antigens remaining, necessary for tumor cell recognition. The antigenic phenotype of human epithelial ovarian cancer after chemotherapy treatment is an important issue in planning potential immunotherapeutic strategies for ovarian cancer. Therefore, we compared the antigen expression in tumor specimens obtained during operation for primary epithelial ovarian tumors and tissue biopsies taken during second-look operations after the administration of combination chemotherapy. A total of 60 ovarian tumors, including 44 serous, 3 mucinous, 2 endometrioid, 1 clear cell, 6 mixed, 2 undifferentiated, and 2 granulosa cell tumors, was studied. Frozen sections of the tumor specimens were stained with 15 different monoclonal antibodies by the indirect immunoperoxidase technique and graded semiquantitatively. The results showed only limited differences in antigenic expression in tumor tissue obtained before and after chemotherapy. Because antigen expression was not altered, immunotherapy making use of these antigenic determinants will not be hampered by prior chemotherapy.

Tumor infiltrating cells in human cancer. On the possible role of CD16+ macrophages in antitumor cytotoxicity.

BACKGROUND: To obtain a better understanding of the mechanism underlying different modalities of immunotherapy, we investigated the types of tumor-infiltrating cells present at the tumor site, with special attention to the presence of macrophages. EXPERIMENTAL DESIGN: Frozen sections of carcinomas of the kidney, colon, breast, lung, ovary, and thyroid gland, as well as malignant melanoma were investigated with a panel of monoclonal antibodies against macrophage, T cell and NK cell associated antigens. Both type and pattern of the tumor-infiltrating cells were analyzed. RESULTS: All tumor-infiltrating cells accumulated preferentially in the stromal bands between tumor cells. In all types of tumor, CD11c+, CD14+, CD68+ and alpha-naphthyl-acetate-esterase positive monocytes/macrophages accounted for most tumor-infiltrating cells. Next in frequency were T lymphocytes (CD2+, CD3+, TCR alpha beta +). Only a few B lymphocytes (CD22+), and T cells expressing the T cell receptor gamma delta (TCR gamma delta) were found. Hardly any lymphoid cells with an NK phenotype (CD3-, CD56+) were present in the tumors studied. Large numbers of CD16+ cells were found, which could be identified as macrophages on the basis of their morphology, positive staining with a panel of monocyte/macrophage markers, and the results of double staining with CD11c. CONCLUSIONS: We have demonstrated the presence of a large number of macrophages in the cellular infiltrates of several types of tumors. The largest numbers of CD16+ macrophages were found in renal cancer, melanoma, and colonic-carcinoma. These are the tumors that are most susceptible to immunotherapy with lymphokine activated killer cells, suggesting that these CD16+ macrophages may be involved in antitumor cytotoxicity. Furthermore, these findings suggest that new strategies of immunotherapy aimed at the use of macrophages present in many tumors could be developed.

Immunotherapy in a human ovarian cancer xenograft model with two bispecific monoclonal antibodies: OV-TL 3/CD3 and OC/TR.

The bispecific antibodies (bs-mAbs) OV-TL 3/CD3 and OC/TR (MOv18/CD3) efficiently mediate ovarian tumor cell lysis by cytotoxic T cells and activated peripheral blood lymphocytes (PBL) in vitro. OV-TL 3/CD3 and OC/TR are reactive with tumor-associated antigens on ovarian carcinoma cells (OA3 and CA-MOv18, respectively), and CD3 on activated PBL, bridging both cells and simultaneously inducing activation of the effector cells. In a comparative study we investigated the therapeutic efficacy of OV-TL 3/CD3 and OC/TR by targeting activated PBL with the bs-mAbs against intraperitoneally growing NIH:OVCAR-3 human ovarian carcinoma cells. As they have good tumor localization characteristics, HPLC-purified bispecific F(ab')2 fragments were used to target highly active PHA and IL-2-stimulated PBL effector cells. The efficacy of OV-TL 3/CD3 was compared to OC/TR with respect to tumor-associated antigen (TAA) binding on NIH:OVCAR-3 ascites cells and NIH:OVCAR-3 tumor cell lysis in vitro. In this report we show that ip ovarian cancer-bearing nude mice treated with IL-2 and activated PBL coated with bispecific F(ab')2 had a significantly longer survival than the untreated mice. No significant difference in survival was found between the OC/TR or OV-TL 3/CD3 bispecific antibody, although MOv18 expression was higher on NIH:OVCAR-3 ascites cells and PBL targeted with OC/TR induced slightly higher tumor cell lysis in vitro. Thus, the therapeutic efficacy of these bs-mAbs in vivo could not be predicted by TAA expression or bs-mAb-mediated tumor cell lysis in vitro.

Association between HLA-expression and infiltration of immune cells in cervical carcinoma.

BACKGROUND: To determine the degree of specificity of the cellular immune response in cervical carcinoma, that is known to be human papillomavirus-related, we investigated the exact relationship between in situ tumor-infiltrating immune cells and the monomorphic/allele-specific HLA expression on the tumor cells. EXPERIMENTAL DESIGN: Attention was focussed on the type, location and number of in situ immunocompetent cells in malignant cervical tissue (N = 30). Immune cell distribution was quantitatively assessed by morphometry for stromal and tumor tissue separately. These results were related to the degree of expression of monomorphic- and allele-specific HLA I and II antigens on the cervical tumor cells. RESULTS: In monomorphic HLA class I downregulated cervical tumors, a significant decrease in tumor-infiltrating CD8+ T cells was observed. However, allele-specific downregulation of respectively HLA-A2, HLA-A3, HLA-Bw4, and HLA-Bw6, did not correlate significantly with a decrease in tumor-infiltrating immune cells. For HLA class II-positive cervical tumors, HLA-DR expression significantly correlated with an increase in the presence of tumor-infiltrating CD3+/CD4+/CD8+ T cells, CD56+ natural killer cells and CD16+ macrophages. No significant correlations were found between alterations in HLA class I or II expression on the tumor cells and stromal infiltrating immune cells. CONCLUSIONS: Our observations provide in situ immunomorphologic evidence that in cervical carcinoma, de novo expression of HLA class II antigens on the tumor cells resulted in an increase of tumor-infiltrating immune cells. In addition, the tumor-infiltrating CD8+ T lymphocytes correlated with monomorphic HLA class I expression on the tumor cells, which stresses the existence of a HLA-restricted immune response of T lymphocytes in cervical carcinoma. These findings might have implications for the biologic behavior of this disease and have to be taken into account in strategies concerning immunotherapy of cervical carcinoma.

Analysis of production, purification, and cytolytic potential of bi-specific antibodies reactive with ovarian-carcinoma-associated antigens and the T-cell antigen CD3.

OV-TL3 and MOv 18 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might therefore be used to develop therapies for ovarian cancer. The bi-specific MAbs alpha T3/OC2 and alpha OC/TR (both being combinations of MOv18 and alpha CD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOv18/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs alpha T3/OC2 and alpha OC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were highly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma supernatants was analyzed by immunohistochemistry, and by testing bi-specific MAb-mediated cytotoxicity against relevant target cells in the presence of effector cells. Stability testing of bi-specific MAb-producing hybridomas showed that, after sub-cloning, clones stably produced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with protein A and by ion-exchange high-pressure liquid chromatography, depending on the sub-class combination of the bi-specific MAb. The purified bi-specific MAbs were tested for their ability to mediate target-cell lysis with the use of cytotoxic T-cell clones and activated peripheral-blood lymphocytes. The purified alpha T3/OC2, alpha OC/TR, and OV-TL3/CD3 were all able to mediate highly specific lysis of various ovarian-carcinoma cell lines. No correlation was found between the level of antigen expression and bi-specific MAb-mediated cytolysis.

Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumor cell line: growth features and cytogenetics.

The characteristics of 7 newly established ovarian carcinoma cell lines and one granulosa tumor cell line obtained from tumor samples of 7 patients with varying histology of the primary tumor are reported. The cell lines were isolated from 5 serous carcinomas, a mucinous carcinoma, an endometrioid carcinoma and a granulosa cell tumor. All cell lines were passaged at least 25 times and showed stable growth rates. Colony-forming efficiency varied on plastic from 2 to 57% and in agar from 0.01 to 9.30%. The DNA index of the granulosa tumor cell line was diploid, while the ovarian carcinoma cell lines were all aneuploid. In 2 cell lines polyploidisation occurred during culturing. A thorough cytogenetic analysis of 7 cell lines revealed that the granulosa tumor cell line has only minor cytogenetic abnormalities (+5, 22q+). In contrast, the epithelial ovarian-cancer cell lines gave very complex karyotypes with numerous markers and structurally rearranged chromosomes. The chromosomes most often in excess were 15 and 20. Structural rearrangements of chromosomes 1, 3, 7 and 11 were prominent in all ovarian cell lines. In addition, we found changes in chromosomes X, 5, 8 and 13 that have rarely been described before.