Tumor invasion, angiogenesis and metastasis involve secretion of proteolytic enzymes and cell migration into blood vessels. Tumor cells are capable of degrading the extracellular matrix via a proteolytic cascade that includes urokinase-type plasminogen activator (uPA) and matrix metalloproteases (MMPs). We have investigated the antitumor and antiangiogenic properties of soy isoflavone genistein in B16 melanoma and F3II mammary carcinoma mouse models. At non-cytotoxic concentrations (0.1-50 μM) genistein induced dosedependent spindle-cell morphology and significantly reduced motility in both cell lines. Genistein inhibited uPA secreted by F3II cell monolayers, while inducing an increase in the proteolytic activity of B16 cells. On the contrary, the compound did not modify the MMP-9 and-2 produced by tumor cells. In vivo, i.p. administration of genistein at a dose of 10 mg/kg/ day reduced tumor-induced angiogenesis in syngeneic mice implanted with B16 or F3II cells. Similar antiangiogenic effects were obtained with a soybean-based diet. This data suggest that tumor cell migration and proteolysis may be associated with the antitumor and antiangiogenic activity of soy isoflavone genistein.

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IMPORTANCE Disseminated retinoblastoma is usually fatal. Identification of small amounts (minimal dissemination [MD]) of tumor cells in extraocular sites might be a tool for designing appropriate treatments. OBJECTIVE To test cone-rod homeobox (CRX) transcription factor as a lineage-specific molecular marker for metastatic retinoblastoma and for evaluation of MD. DESIGN, SETTING, AND PARTICIPANTS In a prospective cohort design study, we evaluated CRX messenger RNA (mRNA) by retrotranscription followed by real-time polymerase chain reaction as a diagnostic test in samples obtained from bone marrow, peripheral blood, and cerebrospinal fluid (CSF) at diagnosis, after induction chemotherapy, and during follow-up. The study was conducted from June 30, 2008, to June 30, 2014. Seventeen retinoblastoma primary tumors, 2 retinoblastoma cell lines, and 47 samples of bone marrow from other cancers (controls) were studied. Seventeen patients with metastatic retinoblastoma (9 at diagnosis, 8 at relapse; age range: 18-41 months) were included. MAIN OUTCOMES AND MEASURES Detection of CRX mRNA as a marker for metastatic retinoblastoma and MD in bone marrow and CSF and its correlation with clinical findings. RESULTS Cone-rod homeobox mRNA was expressed in all tumors (relative expression levels range, 8.1 × 10 −5 to 5.6) and cell lines. In control samples, there was no amplification of CRX; only the housekeeping gene (GAPDH) demonstrated amplification. Bone marrow metastatic cells showed expression of CRX mRNA in all 9 children presenting with metastasis at the diagnosis (relative expression levels, 6.0 × 10 −5 to 0.67). After induction chemotherapy, no evidence of MD of tumor cells was seen in any of the 8 responding children since only GAPDH showed amplification. In the CSF of children who had a metastatic relapse, CRX mRNA detection was positive in 2 patients in whom no conclusive results were reached by immunocytology for disialoganglioside GD2. Minimal dissemination in the CSF was associated with a clinical relapse in 2 cases. No concomitant MD was evident in the bone marrow in any case. CONCLUSIONS AND RELEVANCE These data suggest that CRX mRNA is a novel marker for retinoblastoma at extraocular sites. In this study among patients with bone marrow metastasis, there was a quick, complete, and sustained molecular response after induction chemotherapy. In all patients with secondary metastasis, CSF relapse occurred independently from the bone marrow, suggesting a sanctuary site.

The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreaction was evidenced in normal tissues. The reactivity of 14F7 was detected in all adenocarcinomas of the stomach (12/12), colon (12/12), and pancreas (11/11). A finely granular immunorecognition in esophageal tumors (5/15), epidermoid carcinoma of the rectum (5/7), and basaloid carcinoma (4/5) of the latter as well as in hepatocellular carcinoma (13/14) was also observed. Our results are in agreement with the assumption that NeuGcGM3 ganglioside may be considered as target for passive and active immunotherapy in digestive system malignancies expressing this molecule.