Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy.

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.

Sun Valley summary. Consensus and action items Expedition Inspiration meeting 2000.

The fourth Expedition Inspiration conference was held March 21-23, 2000. While there are other conferences that concentrate on a particular facet of breast cancer, the design and goals of this conference are unusual. In order to maximize interaction of investigators and clinicians the meetings are small, invited, and private. The participants include both senior and junior physicians and scientists involved in clinical and basic research as well as clinical practice. The meetings serve four purposes: (i) Active discussion among participants who do not usually interact, (ii) Develop consensus as to the state of our knowledge as well as an action plan to stimulate future studies, (iii) Develop collaborative projects among the meeting participants, (iv) Foster new investigations by participants as well as others. This year immunology and the role of lymph nodes in breast cancer were the subject of the discussion. Investigators studying breast cancer biology, tumor immunity, potential cancer vaccines, and immunotherapy discussed potential therapeutic manipulation of the immune response to alter the natural history of breast cancer. Regional lymph nodes, the site of the immune response, are often affected early in the spread of breast cancer. For clinicians this involvement is central to planning effective treatment. The paradox that despite the involvement of these lymph nodes in mounting an immune response they are frequently the first site of breast cancer spread stimulated a great deal of discussion and some potentially interesting studies. The following consensus statement is the product of those discussions.

Individual characterisation of the metastatic capacity of human breast carcinoma.

The clinical implications of understanding the invasive and metastatic proclivities of an individual patient's tumour are substantial because the choice of systemic therapy needs to be guided by the likelihood of occult metastasis as well as by knowing when the metastases will become overt. Malignant potential is dynamic, progressing throughout the natural history of a tumour. Required of tumours is the development of critical phenotypic attributes: growth, angiogenesis, invasion and metastagenicity. Characterisation of the extent of tumour progression with regard to these major tumour phenotypes should allow the fashioning of individual therapy for each patient. To examine the clinical parameters and molecularly characterise the metastatic proclivity we have been studying a series of regionally treated breast cancer patients who received no systemic therapy and have long follow-up. Clinically we describe two parameters: metastagenicity - the metastatic proclivity of a tumour, and virulence--the rate at which these metastases appear. Both attributes increase with tumour size and nodal involvement. However, within each clinical group there is a cured population, even in those with extensive nodal involvement, underscoring the heterogeneity of breast cancers within each group and the need for further molecular characterisation. Using biomarkers that characterise the malignant phenotype we have determined that there is progression in the phenotypic changes. Angiogenesis and loss of nm23 are earlier events than the loss of E-cadherin, or abnormalities in TP53. The strongest biomarkers of poor prognosis are p53 and E-cadherin, but even when both are abnormal 42% of node-negative patients are cured indicating that other determinative steps need to occur before successful metastases are established. Identification of these critical later events will further increase the efficacy of determining the malignant capacities of individual tumours.

Separating favorable from unfavorable prognostic markers in breast cancer: the role of E-cadherin.

Distant metastases are the major cause of morbidity and mortality in women with breast cancer. The ability to predict the metastatic proclivity is essential in choosing the optimal treatment. Tumor size and grade, which are frequently used markers in node-negative breast cancer patients, are inadequate markers for prognosis and individualized treatment design. The steps in metastatic progression include angiogenesis, invasion, and changes in adhesion characteristics. We developed a strategy for choosing biomarkers representing these steps in malignant progression to identify patients with occult metastases who will need chemotherapy and spare those women whose tumors have not developed the capacity to spread. To evaluate the added significance of E-cadherin to that of nm23-H1 and angiogenesis in determining metastatic proclivity, we used archival material from 168 node-negative breast cancer patients who were treated with mastectomy without any adjuvant chemotherapy or hormone therapy. Immunohistochemistry was used to detect E-cadherin and nm23-H1 expression, whereas angiogenesis was determined by microvessel count (MVC) after immunohistochemical staining. The median follow-up is 14 years. We found that E-cadherin is better in identifying the poor prognosis patients. The 14-year disease-free survival (DFS) is 84%, 80%, and 56% in patients with high, intermediate, and low E-cadherin. The worst prognosis group using nm23-H1 and MVC as biomarkers has a 14-year DFS of 62%. In this group, if E-cadherin is low, the 14-year DFS is further decreased to 44%. Nm23-H1 and MVC are better in identifying the good prognosis patients. The long-term DFS is >90% if MVC is low or if nm23-H1 is high. Multivariate analysis shows that E-cadherin, nm23-H1, and MVC are more significant prognostic biomarkers than tumor size or grade. Loss of E-cadherin appears to be a latter step in the metastatic progression compared to angiogenesis and the loss of nm23-H1 expression.

Clinical progression of breast cancer malignant behavior: what to expect and when to expect it.

PURPOSE: Seemingly localized breast cancer is a heterogeneous mix of truly localized cancers and cancers with occult metastases. Our purpose is to determine the parameters of metastatic proclivity for the different clinical presentations of operable breast cancer and to present quantitative prognostic information useful to both doctors and patients. PATIENTS AND METHODS: A series of regionally treated breast cancer patients was analyzed to determine the likelihood and time of the appearance of clinical metastases for different clinical subgroups. Patients operated on at the University of Chicago from 1927 to 1987 for clinically regionally localized breast cancer, who received no systemic therapy as a part of their initial treatment, were included. Overall survival and distant disease-free survival in this mature series are analyzed. RESULTS: Metastagenicity, the metastatic proclivity of a tumor, increases with both tumor size and nodal involvement. This is also true for virulence, which is the rate at which these metastases appear. Each clinical group has a cured population, even those with extensive nodal involvement. A table provides a tool for determining the proportion of risk expended in each clinical group as a function of the distant disease-free survival. Whereas the likelihood of metastasis increases with tumor size and nodal involvement, the time to their appearance decreases. CONCLUSIONS: Breast cancer metastagenicity and virulence are heterogeneous even within clinically similar groups of operable breast cancer patients. Tumor progression is correlated with increasing tumor size and nodal involvement. Markers are needed to identify individual tumor virulence and metastagenicity.

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin.

Angiostatin, a proteolytic fragment of plasminogen, inhibits the growth of primary and metastatic tumors by suppressing angiogenesis. When used in combination with ionizing radiation (IR), angiostatin demonstrates potent antitumor synergism, largely caused by inhibition of the tumor microvasculature. We report here the temporal interaction of angiostatin and IR in Lewis lung carcinoma (LLC) tumors growing in the hind limbs of syngeneic mice. Tumors with an initial mean volume of 510 +/- 151 mm3 were treated with IR alone (20 Gy x 2 doses on days 0 and 1), angiostatin alone (25 mg/kg/day divided twice daily) on days 0 through 13, or a combination of the two as follows: (a) IR plus angiostatin (days 0 through 13); (b) IR plus angiostatin (days 0 and 1); and (c) IR followed by angiostatin beginning on the day after IR completion and given daily thereafter (days 2 through 13). By day 14, tumors in untreated control mice had grown to 6110 +/- 582 mm3, whereas in mice treated with: (a) IR alone, tumors had grown to 2854 +/- 338 mm3 (P < 0.05 compared with untreated controls); and (b) angiostatin alone, tumors had grown to 3666 +/- 453 mm3 (P < 0.05 compared with untreated controls). In combined-treatment groups, in mice treated with: (a) IR plus longer-course angiostatin, tumors reached 2022 +/- 282 mm3 (P = 0.036 compared with IR alone); (b) IR followed by angiostatin, tumors reached 2677 +/- 469 mm3 (P > 0.05 compared with IR alone); and (c) IR plus short-course angiostatin, tumors reached 1032 +/- 78 mm3 (P < 0.001 compared with IR alone). These findings demonstrate that the efficacy of experimental radiation therapy is potentiated by brief concomitant exposure of the tumor vasculature to angiostatin.

Monetary reinforcement and Wisconsin Card Sorting performance in schizophrenia: why show me the money?

The Wisconsin Card Sorting Test (WCST) is a measure of concept formation and cognitive flexibility that has been associated with the integrity of the dorsolateral prefrontal cortex. Although patients show deficits on the WCST, training techniques that rely on enhanced instruction are often effective at improving performance, at least temporarily. The beneficial effects of monetary reinforcement alone, however, have not shown such clear-cut effects. Thirty-two schizophrenic inpatients were initially administered a computerized version of the WCST according to standard instructions and then assigned to one of four groups that differed by type of intervention. The level of reinforcement (high vs. low) and enhanced instruction (present vs. absent) were manipulated across the four groups. All patients received a repeat standard administration of the WCST at a 1-week follow-up. Although enhanced instruction showed an initial effect, performance gains fell off at the 1-week retest and approached baseline levels of performance. The level of reinforcement did not make a significant difference. The results indicate that the addition of enhanced verbal instruction yields a benefit, but that contingent monetary reinforcement does not. It appears that deficits on this test are not easily remediated by incentive manipulations.

Aging, progression, and phenotype in breast cancer.

The malignant potential of cancer is dynamic, changing throughout the natural history of a tumor. For breast cancer, this is especially important because the clinical presentation has been altered by the increasing use of screening mammography. The varied outcomes of similarly staged patients is most consistent with breast cancer not being a homogeneous disease, but rather a spectrum of disease states that have varying capacities for growth and metastasis. Evolutionary pressures are at play in both tumor development and during the clinically apparent portion of the life of a tumor and are responsible for this spectrum of tumor heterogeneity. Required of tumors is the development of critical phenotypic attributes: growth, invasion, metastagenicity, and angiogenesis. The combination and permutation of genetic changes that result in the acquisition of these characteristics may vary, but they must result in some expression of each of these phenotypes. The expression of these attributes will differ as tumors evolve to become more adept at each of these characteristics. Recognizing tumor heterogeneity emphasizes the need to determine an individual tumor's place in the evolutionary spectrum. This may be accomplished using clinical features such as size, nuclear grade, and patient age, as well as by examining markers of angiogenesis, metastatic capacity, and proliferation. Identification of the extent of tumor progression with regard to these major tumor phenotypes should allow individual therapy to be fashioned for each patient.

The relationship between nm23, angiogenesis, and the metastatic proclivity of node-negative breast cancer.

Distant metastases are the major cause of morbidity and mortality in women with breast cancer. The prediction of this metastatic proclivity is essential in determining prognosis and should allow an appropriate choice of therapy. A critical look at the metastatic process and its phenotypic expression offers an opportunity to identify some of the important events in the process that may relate to prognosis, with the goal of identifying those patients with occult metastases and also sparing systemic treatment in those patients whose tumors have not developed the capacity for distant spread. To evaluate the significance of nm23 and angiogenesis in the metastatic cascade, we used archival material from 163 node-negative breast cancer patients who had a median follow-up of 14 years. All patients underwent mastectomy and received no adjuvant chemotherapy or hormone or radiation therapy. Immunohistochemistry was used to detect nm23-H1 expression, whereas angiogenesis was determined by microvessel count (MVC). We found the 15-year disease-free survival (DFS) to be significantly better in patients with high nm23 compared with low nm23 (91% compared with 70%, P = 0.008). Low MVC is associated with excellent (92%) long-term DFS. In those patients with high MVC, high nm23 allows the identification of a subgroup with significantly higher DFS (90% compared with 66%, P = 0.02). Among high nuclear grade tumors, if nm23 is high, the DFS is significantly better (89% compared with 68%, P = 0.03). Thus, nm23 is still associated with excellent survival, even when there is unfavorable angiogenesis or nuclear grade. Multivariate analysis confirms that nm23 and MVC are important prognostic factors. High MVC appears necessary but not sufficient for metastasis to occur, whereas low nm23 may further contribute to metastatic progression. Both nm23 and MVC contribute valuable information in characterizing the malignant phenotype.