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Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.

Although biological therapy has shown promising clinical responses in many cancers including metastatic melanoma, only a subset of patients has shown marked regression of lesions. In most patients, systemic administration of biological therapies with cytokines is associated with severe toxicities. Schwager et al., in this issue of Journal of Investigative Dermatology, have examined the role of immunocytokines L19-IL2 and L19-TNF to minimize toxicities, and in combination with Ipilimumab they report complete regression of tumors using syngeneic mouse models. The results, if confirmed in clinical trials, will have major implications for the treatment of human cancers, including melanomas.

Using 22R-hydroxycholesterol as a sub-structure to screen natural compound databases, we identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28days after treatment. In vivo, infusion of sc-7 for 2weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human alpha3 chain, a noncollagenous domain of type IV collagen [alpha3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of alpha3(IV)NC1 to alphaVbeta3 and alpha3beta1 integrins. alpha3(IV)NC1 binds alphaVbeta3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of alpha3beta1 and alphaVbeta3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on alpha3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by alpha3(IV)NC1 in endothelial cells, only alpha3beta1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of alpha3(IV)NC1 to alpha3beta1 integrin leads to inhibition of COX-2-mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IkappaBalpha/NFkappaB axis, and is independent of alphaVbeta3 integrin. Furthermore, beta3 integrin-null endothelial cells, when treated with alpha3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in alpha3 integrin-null endothelial cells, indicating that regulation of COX-2 by alpha3(IV)NC1 is mediated by integrin alpha3beta1. Our in vitro and in vivo findings demonstrate that alpha3beta1 integrin is critical for alpha3(IV)NC1-mediated inhibition of COX-2-dependent angiogenic signaling and inhibition of tumor progression.

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P =.003,.003, and.002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion protein, a mixture of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T lymphocytes, natural killer cells, and macrophages, as well as increased interferon-gamma (IFN-gamma) accumulation, in tumors from animals treated with L19-IL-2 compared to tumors from control groups. The fact that ED-B is 100% homologous in human and mouse, thus ensuring that L19 reacts equally well with human and murine antigen, should ultimately expedite transfer of this reagent to clinical trials.

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.

Background: Sparse data are available with regard to the incidence, clinical characteristics, therapeutic management and prognosis of male patients with germ cell tumors, who relapse more than two years after completion of cisplatin-based chemotherapy., Patients and Methods: A review of 530 patients treated at two institutions from 1978 to April 1994 was conducted. Twenty-five cases of late relapse were identified. Cumulative risk of late relapse was calculated according to the Kaplan-Meier method., Results: 418 of 523 patients (80%) who received their first-line treatment at our institutions were relapse-free at two years. Among these 418 patients 18 cases (4.3%) developed a late relapse. The cumulative risk of late relapse was 1.1% at five years and 4.0% at ten years excluding patients with prior early relapses who carried risks of 9.4% and 29%, respectively (P < 0.0001). No case of late relapse was observed among patients receiving adjuvant chemotherapy. The risk of late relapse was lower in patients with good-risk non-seminomatous germ cell tumors than in poor-risk patients according to Medical Research Council criteria (P < 0.01). Seven further patients were referred from other institutions for treatment of late relapse. At a median follow-up of 38 months (range, 3 to 121) after treatment of late relapse 9 of 25 patients (36%) are continuously disease-free. Six of these nine patients had surgical resection of carcinoma or teratoma as a component of their therapy., Conclusion: The incidence of late relapse after cisplatin-based chemotherapy of germ cell tumors is related to initial tumor burden and is somewhat higher than previously expected. Chemotherapy seems to have only minor curative potential, but localized resectable disease can be cured by surgery. Annual follow-up evaluations allow to detect the majority of late relapses at an asymptomatic stage and should be extended throughout the patient's life.

Bone cells involved in the replacement of cartilage by bone in the endochondral ossification process are known to enter via the medullar pathway. A hypothesis for the development of osteoblasts from chondroblasts was investigated by analyzing the phenotypic characteristics of the 3/A/1D-1M cell line derived from endochondral bone ossicle which was formed after in vivo transplantation of 3/A/1D-1 chondroprogenitor mouse teratocarcinoma cells. The 3/A/1D-1M cell cultures exhibited a triphasic evolution: after reaching confluence (day 3), cultures developed well-delimited cell clusters (days 6-8), which ultimately were organized into multilayered nodules (days 12-15). Electron-microscopic examination of such nodules at day 18 showed the presence of needle-shaped crystals associated with collagen fibrils in the extracellular space. The kinetics of collagen expression, investigated by an immunofluorescence staining procedure showed that, while confluent cultures mainly expressed type III collagen (70% of cells) with some type I (30-40% of cells) and V (30-40% of cells), the type I collagen became the major isoform beginning with day 6. From day 6 onwards, NP40-extracted alkaline phosphatase (AP) activity appeared concomitantly to cell cluster formation, and reached 160 nmol/min/mg of protein at the stage of nodule maturation (day 15). The strong inhibition of enzymatic activity by levamisole and L-homoarginine (IC50 = 0.9 microM and 5 mM, respectively) and its rapid heat inactivation at 56 degrees C (IT50 = 90 s), revealed the bone specificity of AP expressed by 3/A/1D-1M cells. In confluent cultures, brief exposure to parathyroid hormone (10 nM), known to be a bone-resorbing agent, showed a 60% increase in the intracellular cAMP level. In addition, while producing mRNA for the bone-specific protein osteocalcin, 3/A/1D-1M cells also produced type II procollagen mRNA, known to be the major cartilage-related characteristic. This in vitro study demonstrates that the 3/A/1D-1M clonal cell line, originating from 3/A/1D-1 chondroprogenitor cells after in vivo passage, was able to develop differentiated osteoblastic properties as well as the residual expression of the major chondrocytic RNA messenger.

All patients presenting with metastatic teratoma should be regarded as potentially curable and this case demonstrates the multiple treatment modalities which are often needed in the management of such patients. Increasing experience with cisplatin based combination chemotherapy has led to the development of prognostic factors which are used to determine the intensity of treatment given to individual patients. Surgical intervention plays a very important role in the management of residual disease at the completion of chemotherapy. Recognition of the growing teratoma syndrome and the importance of early surgical excision is illustrated by this case. Isolated CNS relapse may occur because the CNS may act as a sanctuary site in patients receiving systemic chemotherapy, but does not preclude long term disease free survival.