Alexandra C. Budinsky

Soluble HER-2/neu neutralizes biologic effects of anti-HER-2/neu antibody on breast cancer cells in vitro.

Amplification and over‐expression of the HER‐2/neu proto‐oncogene are associated with poor prognosis in women with both node‐positive and node‐negative breast cancer. Therefore, the encoded surface glycoprotein represents an attractive target for cancer immunotherapies. Furthermore, the extracellular domain of HER‐2/neu is released from the cell surface by proteolytic cleavage. In the present experiments, we investigated the potential biologic effects of soluble HER‐2/neu with particular emphasis on its interaction with anti‐HER‐2/neu antibodies. A monoclonal antibody specific for the extracellular domain of HER‐2/neu dose dependently inhibited the proliferation of highly HER‐2/neu‐expressing SK‐BR‐3 and BT‐474 breast cancer cells but had no effect on the proliferation of weakly to moderately HER‐2/neu‐expressing MCF‐7, HBL‐100 and ZR‐75‐1 breast cells. Addition of SK‐BR‐3 or BT‐474 cell supernatants with high concentrations of soluble HER‐2/neu led to a neutralization of anti‐HER‐2/neu antibody–mediated inhibition of proliferation due to a binding of soluble HER‐2/neu by the antibody, which could be demonstrated by immunoprecipitation. Furthermore, the ability of anti‐HER‐2/neu antibodies to mediate antibody‐dependent cellular cytotoxicity (ADCC) by lymphokine‐activated killer cells was assessed. Cytolysis of SK‐BR‐3 tumor cells was increased significantly in the presence of anti‐HER‐2/neu antibodies. Similar to the proliferation inhibition, ADCC was neutralized by addition of soluble HER‐2/neu‐containing supernatants. Our data suggest that tumors rich in HER‐2/neu might thus escape certain steps of immunologic control by neutralizing biologic activities of anti HER‐2/neu antibodies due to the presence of soluble HER‐2/neu. Int. J. Cancer 73:875–879, 1997.

Daily prickly pear consumption improves platelet function

Prickly pear is traditionally used by Pima Indians as a dietary nutrient against diabetes mellitus. We examined the effect of daily consumption of 250 g in 8 healthy volunteers and 8 patients with mild familial heterozygous hypercholesterolemia on various parameters of platelet function. Beside its action on lipids and lipoproteins, prickly pear consumption significantly reduced the platelet proteins (platelet factor 4 and beta-thromboglobulin), ADP-induced platelet aggregation and improved platelet sensitivity (against PGI2 and PGE1) in volunteers as well as in patients. Also plasma 11-DH-TXB2 and the WU-test showed a significant improvement in both patients and volunteers. In contrast, collagen-induced platelet aggregation and the number of circulating endothelial cells showed a significant response in patients only. No influence of prickly pear ingestion on peripheral platelet count was monitored. The dietary run-in period did not influence any of the parameters of haemostasis examined. No sex difference was seen. Prickly pear may induce at least part of its beneficial actions on the cardiovascular system via decreasing platelet activity and thereby improving haemostatic balance.

Inhibition of proliferation and induction of apoptosis in soft tissue sarcoma cells by interferon-α and retinoids

SummaryUncontrolled proliferation and a defect of apoptosis constitute crucial elements in the development and progression of tumours. Among many other biological response modifiers known to influence these mechanisms, the efficacy of retinoids and interferons in the treatment of various malignant entities is currently matter of discussion. In the present study, we have investigated the effects of 9-cis-retinoic acid (9cRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (tRA) and interferon-α on proliferation and apoptosis of human soft tissue sarcoma (STS) cell lines HTB-82 (rhabdomyosarcoma), HTB-91 (fibrosarcoma), HTB-92 (liposarcoma), HTB-93 (synovial sarcoma) and HTB-94 (chondrosarcoma) in relation to p53 genotype as well as p53 expression. HTB-91, HTB-92 and HTB-94 STS cells exhibited mutant p53, whereas wild-type p53 was found in HTB-93 STS cells, and a normal p53 status in HTB-82 STS cells, carrying a silent point mutation only. Interferon-α, irrespective of p53 status, inhibited the proliferation of all five cell lines dose- and time-dependently. Similarly, 9cRA, 13cRA and tRA decreased the proliferation of HTB-82 and HTB-93 STS cells, whereas the proliferation of p53-mutated HTB-91, HTB-92 and HTB-94 STS cells remained unchanged. Furthermore, only 9cRA and tRA were capable of inducing apoptosis in HTB-82 and HTB-93 STS cells, whereas HTB-91, HTB-92 and HTB-94 STS cells did not undergo apoptosis under the influence of 9cRA or tRA. Retinoic acid receptor (RAR)-α and RAR-β mRNA were not detectable by Northern blot analysis in the five STS cell lines, whereas mRNA for the universal retinoic acid receptor, RAR-γ, was expressed in all STS cell lines indicating that retinoid resistance was not associated with a lack of RAR expression. Apoptosis was not induced by interferon-α or 13cRA in any of the five STS cell lines tested. Our results indicate that within the panel of tested STS cell lines, inhibition of proliferation and induction of apoptosis result from different mechanisms which differ in their dependence upon the presence of intact p53.

Defective antigen presentation resulting from impaired expression of costimulatory molecules in breast cancer

Previous experiments from our laboratory have shown that immune mechanisms aiming at the destruction of tumour cells including the recognition of target cells and their elimination via the expression of intercellular adhesion molecule‐1 (ICAM‐1; CD54), the production of tumour necrosis factor‐α (TNF‐α) by monocytes and appropriate function of lymphocyte subpopulations were defective in breast cancer. Previous observations were extended to assess expression levels and regulatory mechanisms of costimulatory molecules CD54, CD80 and CD86 on monocytes derived from patients with early breast cancer (EBC). In addition, antigen presentation by antigen‐presenting cells (APC) was analyzed within this context. We report that monocytes derived from patients with EBC exhibited significantly decreased expression levels of CD54 (p = 0.0002), CD80 (p = 0.009) and CD 86 (p = 0.002) compared with monocytes derived from healthy females. Simultaneously, lipopolysaccharide (LPS)‐induced TNF‐α production of monocytes was found to be defective in patients with EBC. Finally, T‐cell proliferation in response to tetanus toxoid (TT) was significantly decreased in patients with EBC compared with healthy control females (p < 0.0001). Furthermore, T‐cell proliferation in response to TT‐pulsed APC derived from healthy controls was significantly inhibited in the presence of anti‐CD54 and/or anti‐CD80 antibodies in a dose‐dependent manner, thus corroborating the necessity of the presence of CD54 and CD80 as costimulatory molecules in the present setting. We conclude that monocytes derived from patients with EBC showed a simultaneous defect of expression of CD54 and its regulation via TNF‐α, CD80 and CD86 as well as T‐cell proliferation following exposure to TT‐pulsed APC. Based upon these findings, it is speculated that defects in costimulatory molecule expression might contribute to tolerance of the immune system towards the presence of malignant cells in patients with EBC. Int. J. Cancer 88:239–244, 2000.

Decreased expression of ICAM‐1 and its induction by tumor necrosis factor on breast‐cancer cells in vitro

In order to study adhesion‐molecule expression and its consequences for cellular recognition, the presence of adhesion molecules ICAM‐1, VCAM‐1, VLA‐4, LFA‐1, alpha, LFA‐1 beta, LFA‐3, β1‐integrin and β3‐integrin was studied on specimens from breast tissue by immunohistochemistry and on cells from breast cell lines propagated in vitro. Breast‐cancer tissue and the breast‐cancer cell lines MCF‐7, SK‐BR‐3 and ZR‐75‐1 showed expression of ICAM‐1 and VLA‐4 significantly lower than that of benign breast cells or normal breast epithelium. Of various cytokines tested, including recombinant human (rh) interleukin‐6 (IL‐6), rh tumor necrosis factor alpha (TNF‐α), interleukin 2 (IL‐2), granulocyte/macrophage‐colony‐stimulating‐factor (GM‐CSF), interferon‐alpha (IFN‐α) and interferon‐gamma (IFN‐γ), only TNF was able to re‐induce expression of ICAM‐1 on cells from MCF‐7, SK‐BR‐3 and ZR‐75‐1. Further, the ability of either unstimulated or lymphokine‐stimulated killer (LAK) cells to recognize and lyse native or TNF‐stimulated breast‐cancer cells was studied. Whereas neither unstimulated lymphocytes or LAK cells were able to lyse untreated breast‐cancer cells deficient for ICAM‐1 expression, pre‐treatment of tumor cells with TNF led to increased tumor‐cell lysis. Anti‐ICAM‐1 antibodies, and pre‐treatment of tumor cells with anti‐TNF‐receptor antibodies, abrogated these findings, corroborating their specificity. We thus conclude that the defective expression of ICAM‐1 in our model might constitute a mechanism by which breast‐cancer cells escape immunologic recognition and lysis by appropriate effector cells. Int. J. Cancer 71: 1086‐1090, 1997.

Short‐term rhythmic proliferation of human breast cancer cell lines: surface effects and fractal growth patterns

Kinetic studies of cell proliferation rates shed light on the growth dynamics of cancer. Most such studies are based on measurements of cell numbers that were evaluated in time intervals of about 12 h. Studies of the initial tumour growth with short measuring intervals are rare. This study was therefore designed with 1 h measuring intervals over a 24 h period. Human breast cancer cell lines (ZR‐75‐1, SK‐BR‐3, MCF‐7) and a benign cell line (HBL‐100) were used to study the hourly thymidine uptake as a measure of cells in synthesis. In parallel experiments, the same cell lines were also exposed to tumour necrosis factor alpha (TNF‐α) to explore the effect of an apoptosis‐inducing substance on initial tumour growth kinetics. In time‐evolution plots, there was an oscillation of the labelling index of thymidine uptake for all investigated cell lines, with and without TNF‐α. Based on the results obtained, a mathematical model was developed mimicking the real experiment. To describe the system dynamically a cellular automaton model was studied. The growth kinetics revealed by the simulation were in accordance with our experimental data. Two‐ and three‐dimensional growth simulations of this computer model yielded objects morphologically similar to real images of human breast cancer. Almost identical fractal dimensions of the virtual and real tumours further supported this visual similarity. The cellular automata models could, therefore, be seen as a bridge towards realistic in vivo scenarios. From a clinical point of view, the results obtained may be applicable not only to primary tumours, but even to tumour cell microfoci and small metastases, which are a major concern in early metastasizing tumours such as breast cancer. Copyright

Pilot study with pegylated liposomal doxorubicin for advanced or unresectable hepatocellular carcinoma

We performed a pilot-study on pegylated liposomal doxorubicin (PLD) for advanced hepatocellular carcinoma. Seventeen patients received 40 mg/m2 PLD intravenously every 4 weeks. A clinical benefit response was achieved in 50% (complete remission 7%, minor remission 7%, stable disease 36%). Toxicities were moderate. In view of these encouraging findings, further studies appear warranted.

Sequential Administration of Interferon-γ, GM-CSF, and Interleukin-2 in Patients With Metastatic Renal Cell Carcinoma: Results of a Phase II Trial

Various cytokine combinations have been tested for efficacy in the treatment of metastatic renal cell carcinoma (MRCC). Because several immunologic synergisms between granulocyte-macrophage colony-stimulating-factor (GM-CSF) and interleukin-2 (IL-2) have been demonstrated, this phase II trial was conducted on the efficacy and toxicity of subcutaneous, sequentially administered, interferon-gamma (IFN&ggr;), GM-CSF, and IL-2. Fifty-five consecutive patients with MRCC were treated with 100 &mgr;g recombinant IFN&ggr;1b administered thrice weekly during weeks 1 and 4, followed by 400 &mgr;g GM-CSF on 5 consecutive days during weeks 2 and 5. In weeks 3 and 6, patients received 4.5 MU recombinant IL-2 from days 1 to 4. The treatment was repeated every 8 weeks. Five (10%) of patients experienced an objective response (complete response [CR]: 2%, partial response [PR]: 8%). Fourteen (26%) patients had stable disease with a median duration of 19 months (6–47+). The median overall survival was 12 months (range: 0.3–44 months). No toxicity greater than World Health Organization grade II was observed, with fever (43%) and erythema (43%) being the most frequent side effects. Compared with other phase II trials with IFN&ggr; and IL-2 alone, the addition of GM-CSF failed to improve response or survival in patients with MRCC.

Sequential administration of interferon γ and interleukin-2 in metastatic renal cell carcinoma: results of a phase II trial

Background: Because of the known efficacy of several cytokines in the treatment of advanced renal cell cancer (RCC), we have conducted a phase II trial of the efficacy and toxicity of subcutaneous interferon γ (IFNγ) and interleukin-2 (IL-2). Methods: 63 patients with progressive metastatic RCC were treated with 100 μg recombinant IFNγ1b administered three times weekly during weeks 1 and 2 and with 4.5 MU recombinant IL-2 administered on 4 consecutive days during weeks 3 and 4, every 6 weeks. Results: 11% of patients had an objective response (CR: 3%, PR: 8%), 33% had SD. Toxicity was generally mild. The median duration of remissions (CR + PR) was 9.6 months; the median duration of SD 8 months. A significant survival benefit was evident at a median observation time of 51 months for patients (44%) responding to therapy (P < 0.0001). Conclusions: we conclude that sequential treatment with IFNγ and IL-2 may prolong survival in patients with metastatic RCC responding to therapy.

Anti-Her-2/neu antibody induces apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status.

Anti-Her-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10–20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53.