Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologousTcells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer^associated antigen a-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor g chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1received a dose escalation of Tcells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specificTcells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells.

Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholineproducing enzymes, and uptake of [methyl-14 C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1 H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 F 0.9 and 5.2 F 2.4 nmol/10 6 cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 F 0.93 in EONT cells; average [phosphocholine] was 3-to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to V0.2. Two-dimensional {phosphocholine/total choline, [total choline]} and {glycerophosphocholine/total choline, [total choline]} maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12-to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 F 0.1 nmol/10 6 cells/h), accounting for a consistently elevated (5-to 15-fold) [methyl-14 C]-choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher (f5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up. (Cancer Res 2005; 65(20): 9369-76)

Promising targeted treatments and immunotherapy strategies in oncology and advancements in our understanding of molecular pathways that underpin cancer development have reignited interest in the tumor-associated antigen Folate Receptor alpha (FRα). FRα is a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Its overexpression in tumors such as ovarian, breast and lung cancers, low and restricted distribution in normal tissues, alongside emerging insights into tumor-promoting functions and association of expression with patient prognosis, together render FRα an attractive therapeutic target. In this review, we summarize the role of FRα in cancer development, we consider FRα as a potential diagnostic and prognostic tool, and we discuss different targeted treatment approaches with a specific focus on monoclonal antibodies. Renewed attention to FRα may point to novel individualized treatment approaches to improve the clinical management of patient groups that do not adequately benefit from current conventional therapies.

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide cholinebased imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKα (but not ChoKβ) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A 2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKα mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC byosinthesis and degradation, respectively. Cancer Res; 70(5); 2126-35. ©2010 AACR.

The important role that the FR plays in cancer development and progression has led to the development of FR-targeted therapeutic approaches. To date, the promising data observed in phase II clinical trials have not been confirmed in phase III studies. Accordingly, there is a need for further research in the refinement of patient selection and identification of new therapeutic combinations. In particular, the development of these targeted therapies requires reliable methods to be developed to detect FR-positive tumors in order to help select patients who may benefit from treatment.

Three new monoclonal antibodies (MAbs) (MOv16, MOv18 and MOv19) were raised against human ovarian carcinoma. To obtain more specific reagents than those produced so far, we adopted the following experimental approach which consisted of: the selection of a poorly differentiated ovarian carcinoma which was unreactive with all the MAb previously selected in our laboratory; and the application of a particular immunization protocol. The reactivity of the selected MAbs was studied by solid-phase RIA on live and fixed cells from tumor cell lines and by immunofluorescence on frozen sections from surgical specimens. The MAb MOv16 reacted with 60% of ovarian carcinomas as well as with a high percentage of other carcinomas and with some normal tissues. In contrast, MOv18 and MOv19 appeared to have restricted specificities for ovarian carcinomas and cystadenomas. Reactivity on other carcinomas was only observed in a few cases and no reactivity was found on non-epithelial tumors or normal tissues. Immunoprecipitation experiments indicated that MOv16 recognizes a 48-50-kDA protein, whereas MOv18 and MOv19 both identify a 38-40 kDA glycoprotein band. Cross-competition experiments, together with a double-determinant immunoradiometric assay which uses MOv18 as catcher and MOv19 as tracer, suggested that they recognize different epitopes carried by the same molecule. The affinity constants of MOv18 and MOv19 were estimated to be in the range of 10(8)-10(9) M-1. Taken together, the properties of these antibodies, their restricted ovarian tumor specificities and relative high affinity constants, suggest that they could represent promising tools for in vivo applications.

Axl, a member of the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases, displays an increasingly important role in carcinogenesis. Analysis of 58 cutaneous melanoma lines indicated that Axl was expressed in 38% of them, with significant overrepresentation in NRAS- compared with BRAF-mutated tumors. Axl activation could be induced by autocrine production of its ligand, Gas6, in a significant fraction of Axl-positive tumors. Pearson's correlation analysis on expression data from five data sets of melanoma lines identified several transcripts correlating positively or negatively with Axl. By functionally grouping genes, those inversely correlated were involved in melanocyte development and pigmentation, whereas those positively correlated were involved in motility, invasion, and microenvironment interactions. Accordingly, Axl-positive melanomas did not express microphthalmia transcription factor (MITF) and melanocyte differentiation antigens (MDAs) such as MART-1 and gp100 and possessed a greater in vitro invasive potential compared with Axl-negative ones. Motility, invasivity, and ability to heal a wound or to migrate across an endothelial barrier were inhibited in vitro by Axl knockdown. Pharmacological inhibition of Axl using the selective inhibitor R428 had comparable effects in reducing migration and invasion. These results suggest that targeted inhibition of Axl signaling in the subset of melanomas lacking MITF and MDAs may represent a novel therapeutic strategy.

Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA-reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential.