Repeated intratumoral administration of ONCOS-102 leads to systemic antitumor CD8+ T-cell response and robust cellular and transcriptional immune activation at tumor site in a patient with ovarian cancer.

Adenoviruses are excellent immunotherapeutic agents with a unique ability to prime and boost immune responses. Recombinant adenoviruses cause immunogenic cancer cell death and subsequent release of tumor antigens for antigen presenting cells, resulting in the priming of potent tumor-specific immunity. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We report a case of a 38-year-old female with Stage 3 metastatic micropapillary serous carcinoma of the ovary. She was treated in a Phase I study with a granulocyte-macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in progressive infiltration of CD8+ lymphocytes into the tumor and concomitant systemic induction of several tumor-specific CD8+ T-cell populations. The patient was alive at the latest follow up more than 20 months after initiation of the study.

Hypertension and overall survival in metastatic colorectal cancer patients treated with bevacizumab-containing chemotherapy.

BACKGROUND: Hypertension (HTN) is a common toxicity of anti-VEGF (vascular endothelial growth factor) antibody treatment. It may be a marker of VEGF signalling pathway inhibition and therefore represent a cancer biomarker in metastatic colorectal cancer (mCRC) patients treated with chemotherapy and bevacizumab. METHODS: A total of 101 consecutive patients with mCRC were treated with standard chemotherapy combined with bevacizumab at dose of 2.5 mg kg(-1) per week in a single centre. The median follow-up time of the patients alive was 64 months. Blood pressure was measured before each bevacizumab infusion, and HTN was graded according to common toxicity criteria for adverse events version 3.0. RESULTS: Overall, 57 patients (56%) developed ≥grade 1 HTN (median blood pressure 168/97 mm Hg), whereas 44 (44%) remained normotensive when treated with bevacizumab-containing chemotherapy regimen. Overall response rate was higher among patients with HTN (30 vs 20%; P=0.025). Hypertension was associated with improved progression-free survival (10.5 vs 5.3 months; P=0.008) and overall survival (25.8 vs 11.7 months; P<0.001), and development of HTN within 3 months had an independent, prognostic influence in a multivariate landmark survival analysis together with other known mCRC prognostic factors (P=0.007). There was no association between HTN and development of thromboembolic complications. CONCLUSION: Hypertension may predict outcome of bevacizumab-containing chemotherapy in mCRC. These data require confirmation in prospective studies including pharmacodynamic and pharmacokinetic analyses.

Docetaxel versus docetaxel alternating with gemcitabine as treatments of advanced breast cancer: final analysis of a randomised trial.

BACKGROUND: Alternating administration of docetaxel and gemcitabine might result in improved time-to-treatment failure (TTF) and fewer adverse events compared with single-agent docetaxel as treatment of advanced breast cancer. PATIENTS AND METHODS: Women diagnosed with advanced breast cancer were randomly allocated to receive 3-weekly docetaxel (group D) or 3-weekly docetaxel alternating with 3-weekly gemcitabine (group D/G) until treatment failure as first-line chemotherapy. The primary end point was TTF. RESULTS: Two hundred and thirty-seven subjects were assigned to treatment (group D, 115; group D/G, 122). The median TTF was 5.6 and 6.2 months in groups D and D/G, respectively (hazard ratio 0.85, 95% confidence interval 0.63-1.16; P = 0.31). There was no significant difference in time-to-disease progression, survival, and response rate between the groups. When adverse events were evaluated for the worst toxicity encountered during treatment, there was little difference between the groups, but when they were assessed per cycle, alternating treatment was associated with fewer severe (grade 3 or 4) adverse effects (P = 0.013), and the difference was highly significant for cycles when gemcitabine was administered in group D/G (P < 0.001). CONCLUSION: The alternating regimen was associated with a similar TTF as single-agent docetaxel but with fewer adverse effects during gemcitabine cycles.

Occupational exposure to radiofrequency fields in antenna towers.

Exposure of workers to radiofrequency fields was assessed in two medium-sized antenna towers. Towers had transmitting antennas from different networks, e.g. mobile phone networks, radio and digital TV sub-stations and amateur radio. The levels of radiofrequency fields were measured close to the ladders of the towers. All measured values were below ICNIRP occupational reference levels.

Transforming growth factor beta1 induces apoptosis in normal melanocytes but not in nevus cells grown in type I collagen gel.

We used type I collagen gel cultures to compare the growth requirements of melanocytes and dermal nevus cells. Melanocytes but not nevus cells undergo apoptosis in collagen unless supplied with growth stimulators such as fibroblast growth factor 2. To characterize the mechanism of melanocyte apoptosis in collagen, we tested the effects of transforming growth factor beta1, known to be functionally active in the skin. When picomolar amounts of transforming growth factor beta1 were added to normal melanocytes grown in type I collagen gel, their apoptosis was dramatically accelerated. In contrast, the apoptotic rate of nevus cells and melanoma cells grown under similar conditions was not affected by transforming growth factor beta1. The increased apoptosis of normal melanocytes was effectively counteracted by addition of either neutralizing transforming growth factor beta1 antibodies or fibroblast growth factor 2 to the collagen gel. Interestingly, the background apoptosis of normal melanocytes was also inhibited by transforming growth factor beta1 antibodies. By Western blotting we detected transforming growth factor beta-like immunoreactivity in melanocyte, nevus cell, and melanoma cell lysates. A sensitive bioassay confirmed that their medium contained considerable amounts of heat-activatable growth inhibitory activity that could partly be neutralized by transforming growth factor beta1 antibodies. It is evident that apoptosis of melanocytes grown in type I collagen gel can be mediated by both endogenous and exogenous transforming growth factor beta. We suggest that the balance between inhibitory growth factors such as transforming growth factor beta and stimulatory growth factors like fibroblast growth factor 2 has the potential to regulate the growth, localization, and survival of normal melanocytes also in vivo. The resistance of nevus cells to transforming-growth-factor-beta-mediated apoptosis may facilitate their ability to grow in the dermal compartment of the skin.

FGF expression allows nevus cells to survive in three-dimensional collagen gel under conditions that induce apoptosis in normal human melanocytes.

Melanocytes, the pigment forming cells of the skin, form an almost nonproliferating cell population located to the lowermost part of the epidermis. Normally melanocytes are not found higher in the epidermis or in the dermis. Nevi consist of melanocytes with altered growth characteristics and localization. The common pigmented nevus, a benign skin lesion, develops when melanocytes proliferate in the dermo-epidermal junction or in the dermis. Here we report growth characteristics of in vitro cultured normal human melanocytes and dermal nevus-derived melanocytes. As previously reported, nevus cells have a moderate to high FGF-2 expression level. Here we demonstrate that dermal nevus cells are able to survive in three-dimensional type 1 collagen culture, while normal human melanocytes rapidly undergo apoptosis. Melanocytes also, however, survive in collagen cultures in the presence of exogenous FGF-2. The survival of nevus cells in collagen is suppressed by protamine, an inhibitor of FGF-mediated cell stimulation. The in vivo growth environment of dermal nevus cells consists largely of type I and type III collagens. The results suggest that FGF-2 expression by nevus cells allows them to adapt to grow in the dermis. FGF-2 obviously has importance as a melanocyte survival factor and probably also in the development of malignant melanoma.

Induction of proliferation in isolated guinea pig gastric epithelium during restitution after superficial injury.

Immediate repair of the gastrointestinal epithelium after superficial injury is called restitution. It is based on the migration of the surviving mucoid neck cells over the area of injury. The involvement of growth factors in the process has been recently documented. They are known to enhance the process (ie, EGF, FGF, TGF-beta) and to activate the basolateral Na+-H+-antiport (EGF). They may exert their effect by activating intracellular tyrosine kinases or by inducing chemotaxis. Yet, their precise mechanism of action in the process is unknown. The aim of the present study was to investigate the effect of modulation of the signal transduction pathway on the occurrence of proliferative mucoid neck and foveolar cells in guinea pig gastric epithelium. Therefore guinea pig gastric epithelium was mounted in Ussing chambers in vitro and perfused 4 hr after superficial injury with 1.25 M NaCl. The potential difference over the epithelium and tissue resistance were recorded simultaneously. The tissue was exposed either to cycloheximide, genistein, or to 4-phorbol myristate 13-acetate (PMA) during the 4-hr recovery, and the expression of proliferative cells was assessed by staining the tissue for proliferative cells (Ki-67). The mean proliferative index of tissues subjected to NaCl injury was significantly higher than that of uninjured control tissues after 4 hr of restitution. Inhibition of the signaling pathway with genistein decreased the proliferative index significantly, while its stimulation with phorbol myristate increased it. Both electrophysiologic and morphologic restitution were sensitive to genistein, but not to PMA or cycloheximide. Superficial epithelial injury results in a significantly increased occurrence of proliferative cells in isolated guinea pig gastric epithelium. This endogenous activation of the tissue is sensitive to inhibition by tyrosine kinases and to stimulation by protein kinases. Electrophysiologic and morphologic recovery are also affected by the modulation of the signaling pathway. This suggests that it is involved in the immediate repair process.

FGF-2 inhibits apoptosis in human teratocarcinoma cells during differentiation on collagen substratum.

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24-48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop. In vivo, such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.

Fibroblast growth factor-mediated stimulation of differentiating teratocarcinoma cells: evidence for paracrine growth regulation.

Tera 2 human embryonal carcinoma cells proliferate rapidly in culture but are capable of differentiating into quiescent cells with neuronal features. We have characterized the effects of exogenous and endogenous fibroblast growth factors on the proliferation of differentiating Tera 2 cells. Exogenous basic fibroblast growth factor (bFGF) stimulated DNA synthesis and induced the proliferation-associated antigen Ki 67 in differentiated Tera 2 cells. Heparin-binding growth factors isolated from the undifferentiated cells excerted a similar stimulatory effect on their differentiated derivatives. The functional potential of these endogenous growth factors was further demonstrated by their ability to stimulate plasminogen activator production by capillary endothelial cells. A major part of the growth promoting activity was removed by absorption with immobilized bFGF antibodies. bFGF was also detected in Tera 2 cells by immunoblotting. The production of heparin-binding growth-promoting activity decreased during differentiation. The results demonstrate a potential role for heparin-binding growth factors in the autocrine or paracrine growth regulation of teratocarcinoma cells.

Development of FGF-dependency in human embryonic carcinoma cells after retinoic acid-induced differentiation.

Rapidly growing human teratocarcinoma cells (Tera-2) can be induced to differentiate into quiescent, nontumorigenic cells expressing neuronal markers. To more closely mimic the in vivo conditions for tumor growth, we grew Tera-2 cells in three-dimensional collagen gel cultures. The undifferentiated cells proliferated in the gel, forming tight colonies. Addition of soluble fibroblast growth factor 1 or 2 (FGF1 or FGF2) into the gel resulted in scattering of single cells throughout the collagen gel. In a FGF gradient the cells moved rapidly toward a higher concentration. On the contrary, cells first differentiated for 8 days in retinoic acid died within a few days after transfer into the collagen gel. Alternatively, if retinoic acid was included in the collagen gel, the proliferating undifferentiated cells died after 4-5 days in the gel. This differentiation-related cell death was completely opposed by including FGF in the collagen gel. When placed in the FGF gradient, the fully differentiated cells survived at the areas of higher FGF concentration, but no more migrated. The survival of retinoic acid-differentiated Tera-2 cells in collagen was also mediated by direct contact with glioma cells or the heparan sulfate-rich portion of glioma or endothelial cell matrix. These effects on differentiated cells were sensitive to inhibition by affinity-purified anti-FGF2 IgG. Thus, FGF has the potential to act as a migration-inducing factor either in solution or, more likely, in vivo, as an immobilized, matrix-bound growth factor directing the movement of responsive cells. The development of differentiation-associated FGF dependency allows survival of the cells only at places where they are in close contact with either FGF-synthesizing cells or FGF-rich extracellular structures such as basement membranes.

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.