FAST: a randomised phase II study of zolbetuximab (IMAB362) plus EOX versus EOX alone for first-line treatment of advanced CLDN18.2-positive gastric and gastro-oesophageal adenocarcinoma.

BACKGROUND: Claudin 18.2 (CLDN18.2) is contained within normal gastric mucosa epithelial tight junctions; upon malignant transformation, CLDN18.2 epitopes become exposed. Zolbetuximab, a chimeric monoclonal antibody, mediates specific killing of CLDN18.2-positive cells through immune effector mechanisms. PATIENTS AND METHODS: The FAST study enrolled advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients (aged ≥18 years) with moderate-to-strong CLDN18.2 expression in ≥40% tumour cells. Patients received first-line epirubicin + oxaliplatin + capecitabine (EOX, arm 1, n = 84) every 3 weeks (Q3W), or zolbetuximab + EOX (loading dose, 800 mg/m2 then 600 mg/m2 Q3W) (arm 2, n = 77). Arm 3 (exploratory) was added after enrolment initiation (zolbetuximab + EOX 1000 mg/m2 Q3W, n = 85). The primary endpoint was progression-free survival (PFS) and overall survival (OS) was a secondary endpoint. RESULTS: In the overall population, both PFS [hazard ratio (HR) = 0.44; 95% confidence interval (CI), 0.29-0.67; P < 0.0005] and OS (HR = 0.55; 95% CI, 0.39-0.77; P < 0.0005) were significantly improved with zolbetuximab + EOX (arm 2) compared with EOX alone (arm 1). This significant PFS benefit was retained in patients with moderate-to-strong CLDN18.2 expression in ≥70% of tumour cells (HR = 0.38; 95% CI, 0.23-0.62; P < 0.0005). Significant improvement in PFS was also reported in the overall population of arm 3 versus arm 1 (HR = 0.58; 95% CI, 0.39-0.85; P = 0.0114) but not in high CLDN18.2-expressing patients; no significant improvement in OS was observed in either population. Most adverse events (AEs) related to zolbetuximab + EOX (nausea, vomiting, neutropenia, anaemia) were grade 1-2. Grade ≥3 AEs showed no substantial increases overall (zolbetuximab + EOX versus EOX alone). CONCLUSIONS: In advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients expressing CLDN18.2, adding zolbetuximab to first-line EOX provided longer PFS and OS versus EOX alone. Zolbetuximab + EOX was generally tolerated and AEs were manageable. Zolbetuximab 800/600 mg/m2 is being evaluated in phase III studies based on clinical benefit observed in the overall population and in patients with moderate-to-strong CLDN18.2 expression in ≥70% of tumour cells.

A multicentre, phase IIa study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophagus: the MONO study.

BACKGROUND: Claudin 18.2 (CLDN18.2) is physiologically confined to gastric mucosa tight junctions; however, upon malignant transformation, perturbations in cell polarity lead to CLDN18.2 epitopes being exposed on the cancer cell surface. The first-in-class monoclonal antibody, zolbetuximab (formerly known as IMAB362), binds to CLDN18.2 and can induce immune-mediated lysis of CLDN18.2-positive cells. PATIENTS AND METHODS: Patients with advanced gastric, gastro-oesophageal junction (GEJ) or oesophageal adenocarcinomas with moderate-to-strong CLDN18.2 expression in ≥50% of tumour cells received zolbetuximab intravenously every 2 weeks for five planned infusions. At least three patients were enrolled in two sequential cohorts (cohort 1300 mg/m2; cohort 2600 mg/m2); additional patients were enrolled into a dose-expansion cohort (cohort 3600 mg/m2). The primary end point was the objective response rate [ORR: complete and partial response (PR)]; secondary end points included clinical benefit [ORR+stable disease (SD)], progression-free survival, safety/tolerability, and zolbetuximab pharmacokinetic profile. RESULTS: From September 2010 to September 2012, 54 patients were enrolled (cohort 1, n = 4; cohort 2, n = 6; cohort 3, n = 44). Three patients in cohort 1 and 25 patients in cohorts 2/3 received at least 5 infusions. Antitumour activity data were available for 43 patients, of whom 4 achieved PR (ORR 9%) and 6 (14%) had SD for a clinical benefit rate of 23%. In a subgroup of patients with moderate-to-high CLDN18.2 expression in ≥70% of tumour cells, ORR was 14% (n = 4/29). Treatment-related adverse events occurred in 81.5% (n = 44/54) patients; nausea (61%), vomiting (50%), and fatigue (22%) were the most frequent. CONCLUSIONS: Zolbetuximab monotherapy was well tolerated and exhibited antitumour activity in patients with CLDN18.2-positive advanced gastric or GEJ adenocarcinomas, with response rates similar to those reported for single-agent targeted agents in gastric/GEJ cancer trials. CLINICALTRIALS.GOV NUMBER: NCT01197885.

Discordant results obtained for different methods of HER-2/neu testing in breast cancer--a question of standardization, automation and timing.

BACKGROUND: HER-2/neu positivity is required for the selection of stage IV breast cancer patients for trastuzumab therapy. We compared the results of the recommended immunohistochemistry (IHC) evaluation with the automated ACIS IHC system and with fluorescence in situ hybridization (FISH). These HER-2/neu tissue results were correlated with the serum HER-2/neu (sHER-2/neu) levels at the time of metastatic spread. PATIENTS AND METHODS: A total of 61 IHC slides from 30 patients were stained using the HercepTest. HER-2/neu gene amplification was determined using the Ventana FISH assay. sHER-2/neu levels were measured with the Oncogene Science" ELISA kit. The concordance of HER-2/neu results was determined using the concordance index Kappa (kappa). RESULTS: The best concordance between any IHC and FISH was found for the automated ACIS system (88.5%, kappa=0.68, category "good"). The comparison between the manual interpretations and the automated IHC was categorized as "very good" (95.1%, kappa=0.85). The median sHER-2/neu level of FISH positive patients was significantly higher (67 ng/mL) than that of FISH negative patients (17 ng/mL, p=0.018). The increase in HER-2/neu positivity comparing tissue to stage IV serum was statistically significant (p=0.001). CONCLUSIONS: The concordance between conventional IHC and computerized analysis was very good. The number of patients with stage IV breast cancer with an elevated sHER-2/neu level was much higher than HER-2/neu positivity in tissue. This discrepancy is only partially explained by the influence of tumor load. Patients with an elevated sHER-2/neu level and no tissue overexpression should be considered for retesting of tissue or a new biopsy.

Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.

To identify novel markers differentially expressed in ovarian cancer versus normal ovary, we hybridized microarrays with cDNAs derived from normal human ovaries and advanced stage ovarian carcinomas. This analysis revealed down-regulation of the caveolin-1 gene (CAV1) in ovarian carcinoma samples. Suppression of CAV1 in ovarian carcinomas was confirmed using a tumor tissue array consisting of 68 cDNA pools from different matched human tumor and normal tissues. Immunohistochemistry demonstrated expression of caveolin-1 in normal and benign ovarian epithelial cells, but loss of expression in serous ovarian carcinomas. In low-grade carcinomas, redistribution of caveolin-1 from a membrane-associated pattern observed in normal epithelium to a cytoplasmic localization pattern was observed. No expression of caveolin-1 was detectable in four of six ovarian carcinoma cell lines investigated. In SKOV-3 and ES-2 carcinoma cells, which express high levels of the caveolin-1 protein, phosphorylation of the 22-kd caveolin-1 isoform was detected. Inhibition of both DNA methylation and histone deacetylation using 5-aza-2'deoxycytidine and Trichostatin A, respectively, relieves down-regulation of caveolin-1 in OAW42 and OVCAR-3 cells which is in part mediated by direct regulation at the mRNA level. Expression of CAV1 in the ovarian carcinoma cell line OVCAR-3, resulted in suppression of tumor cell survival in vitro, suggesting that the CAV1 gene is likely to act as a tumor suppressor gene in human ovarian epithelium.

Diagnostic difficulties before definitive treatment of an extragonadal retroperitoneal germ cell tumor.

A primary extragonadal germ cell tumor of the retroperitoneum was diagnosed in a 47-year-old man without elevated serum alpha-fetoprotein, human chorionic gonadotropin, or lactate dehydrogenase levels. The diagnosis was made by histologic analysis after resection. The patient responded well to a combination of cisplatin, etoposide, and ifosfamide, achieving a partial response with four cycles. Residual tumor resection revealed necrotic tissue only. The patient was alive and disease free 24 months after diagnosis. The diagnostic difficulties of this particular situation are discussed.

Down-regulation of caveolin-1, a candidate tumor suppressor gene, in sarcomas.

Caveolae are plasma membrane microdomains that have been implicated in the regulation of several intracellular signaling pathways. Previous studies suggest that caveolin-1, the main structural protein of caveolae, could function as a tumor suppressor. Caveolin-1 is highly expressed in terminally differentiated mesenchymal cells including adipocytes, endothelial cells, and smooth muscle cells. To study whether caveolin-1 is a possible tumor suppressor in human mesenchymal tumors, we have analyzed the expression using immunohistochemistry in normal mesenchymal tissues, 22 benign and 79 malignant mesenchymal tumors. Caveolin-1 was found to be expressed in fibromatoses, leiomyomas, hemangiomas, and lipomas at high levels comparable to normal mesenchymal tissues. The expression of caveolin-1 was slightly reduced in four of six well-differentiated liposarcomas and strongly reduced or lost in three of three fibrosarcomas, 17 of 20 leiomyosarcomas, 16 of 16 myxoid/round cell/pleomorphic liposarcomas, five of eight angiosarcomas, 15 of 18 malignant fibrous histiocytomas, and eight of eight synovial sarcomas. The immunohistochemical findings were confirmed by Western blot analysis in a number of tumors. High levels of both the 24-kd [alpha]- and the 21-kd [beta]-isoform of caveolin-1 were detected in the nontumorigenic human fibroblast cell line IMR-90. In contrast, in HT-1080 human fibrosarcoma cells, caveolin-1 is strongly down-regulated. We show that the [alpha]-isoform of caveolin-1 is potently up-regulated in HT-1080 cells by inhibition of the mitogen-activated protein kinase-signaling pathway with the specific inhibitor PD 98059, whereas the specific inhibitor of DNA methylation 5-aza-2'-deoxycytidine only marginally up-regulates caveolin-1. In addition, re-expression of caveolin-1 in HT-1080 fibrosarcoma cells potently inhibited colony formation. From these we conclude that caveolin-1 is likely to act as a tumor suppressor gene in human sarcomas.

c-erbB-2 Antisense Oligonucleotides Inhibit Serum-Induced Cell Spreading of Ovarian Cancer Cells.

Amplification or overexpression of the c-erbB-2 oncogene (also known as HER-2, neu) is a frequent event in many types of human cancer including 20-30% of ovarian cancers where it characterizes a group of patients with poor prognosis (1,2). The expression of p185 (c-erbB-2) is in contrast quite restricted in normal adult tissues (3). The c-erbB-2 oncogene product (p185c-erbB-2) is a growth factor receptor (GFR) with extensive homology to the receptor for the epidermal growth factor (EGFR) and the c-erbB-3 and c-erbB-4 gene products (4). According to the hypothesis that c-erbB-2 is involved in pathogenesis and progression of human cancer, the overexpression in fibroblasts leads to the appearance of a transformed phenotype, capable of forming colonies in soft agar and inducing tumors in mice (5).

Suppression of the c-erbB-2 gene product decreases transformation abilities but not the proliferation and secretion of proteases of SK-OV-3 ovarian cancer cells.

The overexpression of the c-erbB-2 oncogene product has been reported in approximately 20-30% of human ovarian cancers and has been correlated with a poor prognosis in ovarian cancer patients. To investigate the function of p185(c-erbB-2) in human ovarian cancer cells, a c-erbB-2-specific single-chain antibody (scFv-5R) was expressed in the c-erbB-2-overexpressing SK-OV-3 cell line using a retroviral expression vector. Eight individual clones expressing the single-chain antibody were isolated. These clones have a prominent retention of the cell surface p185(c-erbB-2). In this study we compared the proliferation rate, the anchorage-independent growth, the secretion of matrix metalloproteases and of the urokinase-type plasminogen activator. The clones expressing the c-erbB-2 single-chain antibody, the control cells harbouring the empty vector and the parental SK-OV-3 cells they all had similar proliferation rates in the presence of 10% serum and secreted similar amounts of matrix metalloproteases and of the urokinase-type plasminogen activator. However, the expression of the c-erbB-2 oncogene product offers a strong growth advantage under serum-reduced conditions with 1% serum. In contrast to the parental SK-OV-3 and empty vector control cells, the scFv-5R-expressing clones were not able to grow anchorage-independently. These findings suggest that c-erbB-2 enhances transformation abilities of SK-OV-3 ovarian cancer cells without affecting the secretion of proteases and the proliferation of SK-OV-3 ovarian cancer cells in the presence of high concentrations of serum.

Involvement of the c-erbB-2 oncogene product in the EGF-induced cell motility of SK-OV-3 ovarian cancer cells.

The epidermal growth factor (EGF) induces the rapid formation of dendritic processes and the dissociation of SK-OV-3 ovarian cancer cells. The SK-OV-3 cell line is characterized by over-expression of the c-erbB-2 oncogene product (p185(c-erbB-2)). To investigate the role of p185(c-erbB-2) in cell motility, a c-erbB-2-specific single-chain antibody was expressed in SK-OV-3 cells using a retroviral expression vector. Eight individual clones expressing the single chain antibody were isolated. These clones have prominent retention of the cell-surface p185(c-erbB-2). The EGF-induced morphologic changes and cell motility of the single-chain-antibody-expressing clones were strongly inhibited, as observed in cell dissociation and in transmigration experiments. However, the suppression of p185(c-erbB-2) does not inhibit the motility signal elicited by 12-O-tetradecanoyl-phorbol-13-acetate. These data indicate that the c-erbB-2 oncogene product is essential to mediate the motility signal of EGF to SK-OV-3 ovarian cancer cells. The enhancement of tumor-cell motility may be partially responsible for the unfavorable prognosis of ovarian cancer with over-expression of c-erbB-2.

Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor I receptor in ovarian cancer cells.

The insulin-like growth factors (IGF-I and IGF-II) play a key role in cellular proliferation and are involved in cellular transformation. The expression of the IGF-I receptor has been demonstrated in a variety of human tumor cell lines including ovarian cancer cells. Phosphorothioate antisense oligodeoxynucleotides (S-ODNs) were analyzed for their potential to suppress the IGF-I receptor in the NIH: OVCAR-3 ovarian cancer cell line. The application of the antisense S-ODN reduced potently the cell growth of unstimulated NIH:OVCAR-3 cells, whereas sense and mismatch S-ODNs were without any effect. This effect resembled that of the monoclonal antibody (MAb) alphaIR-3. In contrast to the antisense compound, this MAb only partially inhibited the IGF-I-induced proliferation of ovarian cancer cells. The concentration of the antisense S-ODN to exhibit an identical inhibition of cell proliferation was reduced to 50 nM when the oligonucleotides were delivered by the cationic lipid formulation lipofectin. The specificity of the antisense S-ODN action was confirmed by reduction of the receptor protein and of the receptor mRNA, as assayed by flow cytometry and by Northern blot hybridizations. Our data demonstrate the potency of antisense S-ODNs to target the IGF-I receptor message and show that antisense strategies against the IGF-I receptor may provide new strategies for the therapy of ovarian cancer.

Selection of a high activity c-erbB-2 ribozyme using a fusion gene of c-erbB-2 and the enhanced green fluorescent protein.

In different types of human cancer there is an overexpression of the c-erbB-2 (HER2/neu) oncogene, which is thought to be involved in tumor progression. Therefore the c-erbB-2 oncogene is an attractive target for tumor-specific gene therapy. In this report we have characterized a hammerhead ribozyme against the c-erbB-2 mRNA with high cleavage activity. To select the optimum sequence, the activity of five hammerhead ribozymes was tested in a cell-free assay. The hammerhead ribozyme recognizing the GUC sequence at position +631 to +633 of the c-erbB-2 mRNA (RZ631) efficiently cleaves in vitro transcribed fragments of the c-erbB-2 mRNA [169 to 1450 nucleotides (nt)] under multiple-turnover conditions. The ribozyme coding sequence was subsequently cloned between the A and the B box promoter sequences of the fowl adenovirus type 1 virus-associated RNA (CELO VA) gene. The in vitro activity of RZ631 was shown to be unaffected by the polymerase III promoter flanking sequences. The ability of RZ631 to inhibit the synthesis of the c-erbB-2 gene product in tumor cells was assayed by cotransfection of the ribozyme with a fusion gene of c-erbB-2 and the gene for the enhanced green fluorescent protein as a reporter. The synthesis of the fluorescent fusion protein in NIH:OVCAR-3 ovarian cancer cells was potently inhibited by RZ631, as assayed by flow cytometry. An antisense control vector, where the catalytic core was replaced by a single base, showed a weaker inhibition of expression of the c-erbB-2 derivative. The results suggest that the inhibitory effect of this c-erbB-2 ribozyme is caused by an antisense effect as well as by an additional ribozyme-mediated increase in inhibition. We conclude that this c-erbB-2 ribozyme in conjunction with a polymerase III-based expression system should be useful for the efficient downregulation of the c-erbB-2 oncogene in ovarian cancer cells.

c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides inhibit growth and serum-induced cell spreading of P185c-erbB-2-overexpressing ovarian carcinoma cells.

Overexpression of the c-erbB-2 proto-oncogene product (p185c-erbB-2) occurs frequently in different types of human cancer and is correlated with a significantly decreased survival in ovarian cancer patients. The effect of c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides (S-ODNs) was examined on the ovarian cancer cell line SK-OV-3. p185c-erbB-2 levels were specifically reduced by a single-dose application of 5 microM c-erbB-2 anti-sense S-ODNs. This was accompanied by a 60% inhibition of anchorage-dependent cell growth. More strikingly, c-erbB-2 anti-sense S-ODNs almost completely abrogated serum-induced cell spreading. A control of complementary sense oligodeoxynucleotides did not show significant inhibitory effects on cell growth or on cell spreading. The inhibition of cell spreading was imitated by a monoclonal antibody (9G6) targeting the extracellular domain of p185c-erbB-2 and by the tyrosine kinase inhibitor erbstatin. The inhibitory activity of these 2 compounds was lost after a few hours, while the inhibition of serum-induced cell spreading by anti-sense S-ODNs was still present after 24 hr. Our results show that c-erbB-2 anti-sense S-ODNs effectively inhibit the mitogenic and spreading activity of p185c-erbB-2 in ovarian cancer cells. Thus, anti-sense strategies have the potential of providing new strategies for the therapy of ovarian cancer.

Two distinct functional effects of protein phosphatase inhibitors on guinea-pig cardiac L-type Ca2+ channels.

1. The effects of the phosphatase inhibitors okadaic acid and calyculin A on single guinea-pig ventricular L-type Ca2+ channels were studied. The inactive derivative norokadaone was used as a negative control. 2. The two known effects of cAMP-dependent stimulation are mimicked by the phosphatase inhibitors to a varying extent. Only okadaic acid promotes the high-activity gating mode ('mode 2'), while calyculin A increases channel availability to a larger extent. As revealed by kinetic analysis of slow gating, the two phosphatase inhibitors retard a slow rate constant, which is assumed to represent exit from the available state by dephosphorylation. Norokadaone was inactive in both regards. 3. Mode 2 gating elicited by very positive prepulses is augmented by okadaic acid, and mode 2 lifetime is prolonged. Calyculin A fails to affect these parameters. Thus, voltage-facilitated mode 2 gating reveals the same pharmacological properties as the mode 2 sweeps observed using conventional pulse protocols. 4. The results are interpreted in terms of the different sensitivity of protein phosphatase subtypes towards the inhibitors: channel availability appears to be controlled by a phosphorylation site dephosphorylated by a type 1-like phosphatase, while mode 2 gating is coupled to a distinct site, dephosphorylated by a type 2A-like phosphatase.

Biochemical and electrophysiological mechanisms of the positive inotropic effect of calyculin A, a protein phosphatase inhibitor.

Calyculin A (CyA; 1 microM) increased the force of contraction in isolated guinea pig papillary muscles to 144% of control without affecting contraction parameters. The effect of CyA on L-type calcium channels was assessed in cell-attached patches of guinea pig ventricular cardiomyocytes. Unitary Ba++ current recordings revealed that CyA at micromolar concentrations enhanced channel availability almost 2-fold, whereas the duration of individual openings and closures remained unchanged. In whole-cell recordings with Ca++ as the charge carrier, intracellular dialysis of 1 microM CyA enhanced peak current to a similar extent. In homogenates from guinea pig ventricles, 1 microM CyA completely inhibited phosphorylase phosphatase activity. In isolated [32P]-labeled guinea pig ventricular cardiomyocytes, 1 microM CyA increased the phosphorylation state of phospholamban (to 267% of control), that of the inhibitory subunit of troponin (to 182% of control) and those of various additional proteins. We conclude that the effects of CyA are likely to be mediated by increasing the phosphorylation state of several regulatory proteins.

Isoelectric focusing pattern of human amniotic fluid alpha-fetoprotein.

Isoelectric focusing (IEF) of amniotic fluid alpha-fetoprotein (AFP) in thin-layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTD), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholinesterase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD).