Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.

Gliomas are the most common intracranial primary tumors, for which very few therapeutic options are available. The most malignant subtype is the glioblastoma, a disease associated with a 5-year survival rate lower than 5%. Given that research in glycobiology continues highlighting the role of glycans in tumor cell biology, it offers an interesting niche for the search of new therapeutic targets. In this study, we characterized aberrant glycosylation and its impact on cell biology over a broad panel of high- and low-grade glioma cell lines. Results show high expression of terminal Lewis glycans, mainly SLex, and overexpression of sialyl- and fucosyltransferases involved in their biosynthesis in high-grade glioma cell lines. Moreover, we report an association of complex multi-antennary N-glycans presenting ß1,6-GlcNAc branches with the high-grade glioma cells, which also overexpressed the gene responsible for these assemblies, MGAT5. In addition, downmodulation of N-glycosylation by treatment with the inhibitors Tunicamycin/Swainsonine or MGAT5 silencing decreased SLex expression, adhesion and migration in high-grade glioma cells. In contrast, no significant changes in these cell capacities were observed in low-grade glioma after treatment with the N-glycosylation inhibitors. Furthermore, inhibition of histone deacetylases by Trichostatin A provoked an increase in the expression of SLex and its biosynthetic related glycosyltransferases in low-grade glioma cells. Our results describe that aggressive glioma cells show high expression of Lewis glycans anchored to complex multi-antennary N-glycans. This glycophenotype plays a key role in malignant cell behavior and is regulated by histone acetylation dependent mechanisms.

Aberrant O-glycosylation modulates aggressiveness in neuroblastoma.

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Antibody-dependent cell-mediated cytotoxicity induced by active immunotherapy based on racotumomab in non-small cell lung cancer patients.

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.

Synergistic potentiation of the anti-metastatic effect of anti EGFR mAb by its combination with immunotherapies targeting the ganglioside NGcGM3.

Several Anti-EGFR mAbs are register for the treatment of human cancer. However, their impact on patients overall survival has been limited by tumor resistance. N-Glycolyl variant of GM3 ganglioside (NGcGM3) is specifically expressed in some human tumors, and it has been associated with a poor prognosis. Several reports have documented that GM3 physically associates to EGFR inhibiting its ligand depend phosphorylation, but it also facilitates an alternative/compensatory signaling cascade mediated by Uroquinase Plasminogen Activator Receptor (uPAR) and integrin α5ß1 interaction. However, the difference between NGc and N-Acetylated (NAc) variants of GM3 regarding such interactions is unknown. We hypothesized that enrichment of NGcGM3 expression in tumors relates to advantages of this ganglioside, on ensuring both EGFR and uPAR pathways optimal function. We explored the impact of combining an anti-EGFR (7A7 mAb) with anti-NGcGM3 therapies: NGcGM3/VSSP vaccine or 14F7 mAb. Both combinations synergistically increase overall survival in two models of lung metastasis: 3LL-D122 and 4T1; but combination with NGcGM3/VSSP vaccine is significantly more effective. In 3LL-D122-metastasis, of mice treated with the best combination, both EGFR and uPAR/α5ß1 integrin pathways are turn off (I.e expression of uPAR/α5ß1; and phosphorylation of EGFR, Stat3, Src and FAK are reduced); and tumor angiogenesis is decreased. Interestingly, combination treatment increases tumor infiltrating CD4+T, CD8+T and NK+-cells. Furthermore, a positive clinical outcome is reported for a cancer patient treated with an anti-EGFR mAb and anti-NGcGM3 therapy. Overall, our results support the combination of anti EGFR antibodies with therapies targeting NGcGM3 to increase their efficacy in future clinical trials.

Minimal Disseminated Disease in Nonmetastatic Retinoblastoma With High-Risk Pathologic Features and Association With Disease-Free Survival.

IMPORTANCE: Fatal metastatic relapse may occur in children with retinoblastoma and high-risk pathologic features (HRPFs). Minimal dissemination (MD) may be an additional tool for risk estimation. The use of cone-rod homeobox (CRX) transcription factor messenger RNA for MD evaluation in metastatic retinoblastoma was previously reported, but no data in nonmetastatic cases with HRPFs are available. OBJECTIVES: To evaluate whether MD is detectable in patients with nonmetastatic retinoblastoma and to assess its prognostic effect on disease-free survival (DFS). DESIGN, SETTING, AND PARTICIPANTS: This single-institution cohort study of patients with nonmetastatic retinoblastoma and HRPFs used prospectively defined inclusion criteria and a sampling strategy to procure bone marrow (BM) and cerebrospinal fluid (CSF) samples from May 1, 2007, through October 31, 2013. Median follow-up was 38 months (range, 8-89 months). Survival analysis was closed in December 2015, and no further updates were made after that point. INTERVENTIONS: The study evaluated CRX messenger RNA by quantitative polymerase chain reaction in BM and CSF at diagnosis and follow-up. In 14 patients, GD2 synthase was used instead of CRX for CSF evaluation. Patients were treated under uniform guidelines. MAIN OUTCOMES AND MEASURES: Metastatic relapse. RESULTS: The study included 96 children (median age at study inclusion, 26 months; range, 1-168 months; 46 male [47.9%]; 50 female [52.1%]) with nonmetastatic retinoblastoma and HRPFs (isolated massive choroidal invasion in 14, postlaminar optic nerve invasion in 51 [26 with concomitant massive choroidal and 13 with scleral invasion], 12 with scleral invasion without postlaminar optic nerve invasion, and 7 with tumor at the resection margin of the optic nerve) were evaluated at the time of primary or secondary enucleation. Minimal dissemination was detected in 9 patients (7 BM samples and 2 CSF samples) and was associated with extension beyond the resection margin of the optic nerve and scleral involvement, but only the former was independently associated (adjusted odds ratio, 57.0; 95% CI, 4.8-678.2; P = .001). In addition, MD occurred in 8 of the 43 International Intraocular Retinoblastoma Classification group E eyes with glaucoma (18.6%) and in 8 of 80 (10%) and 1 of 16 children (6.3%) who underwent primary or secondary enucleation, respectively. Children with MD had a 3-year DFS of 0.78 compared with 0.98 in those without MD (95% CI for the difference in DFS, 0.17-0.23; P = .004). CONCLUSIONS AND RELEVANCE: These findings identified a high-risk population of children with retinoblastoma and HRPFs with MD. Because the number of events was small, these results, which suggest that children with International Intraocular Retinoblastoma Classification group E retinoblastoma and glaucoma have a higher risk of MD at diagnosis, should not be considered definitive at this time.

Comparability of Antibody-Mediated Cell Killing Activity Between a Proposed Biosimilar RTXM83 and the Originator Rituximab.

BACKGROUND: Biosimilars are described as biological products that resemble the structure of original biologic therapeutic products, with no clinically meaningful differences in terms of safety and effectiveness from the original. A wide range of biosimilars are under development or are already licensed in many countries. Biosimilars are earning acceptance and becoming a reality for immunotherapy treatments mainly based on the alternatives for the commercial anti-CD20 monoclonal antibody rituximab. The most important mechanism of action reported for this antibody is the induction of antibody-dependent cell cytotoxicity (ADCC), which is associated with the polymorphisms present at the 158 position in the IgG receptor FcγRIIIa. OBJECTIVE: The aim of the study was to validate the functional comparability between the proposed rituximab biosimilar RTXM83 and the original product. To achieve this we assessed the binding capacity and ADCC induction of this biosimilar, taking into account the different FcγRIIIa-158 polymorphisms. METHODS: Binding capacity was evaluated by flow cytometry using CD20 positive cells and a wide range of antibody concentrations. The FcγRIIIa-158 polymorphisms were analyzed by polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion. ADCC was measured by a colorimetric lactate dehydrogenase-release assay, using effector cells from donors with different FcγRIIIa-158 polymorphisms. RESULTS: Binding capacity assay showed no differences between both products. Regarding ADCC, a similar relative potency was obtained between both antibodies, showing a higher response for the FcγRIIIa-158 valine/valine (V/V) polymorphism compared to the phenylalanine/phenylalanine (F/F), for both rituximab and RTXM83. CONCLUSION: Our data strongly suggest the biocomparability between the proposed biosimilar and the originator rituximab, in antibody recognition and ADCC activity. Additionally, our results suggest that donors with the FcγRIIIa-158V/V polymorphism induce a higher ADCC response, as has been reported.

Racotumomab for treating lung cancer and pediatric refractory malignancies.

INTRODUCTION: Racotumomab (originally known as 1E10 mAb) is an anti-idiotype murine IgG1 directed to membrane glycoconjugates expressed in aggressive solid tumors. It was developed as a mirror image of the idiotype of another antibody against N-glycolyl-containing molecules, such as the NeuGcGM3 ganglioside. After a successful phase II/III study, racotumomab formulated in alum was conditionally approved in Latin American countries as maintenance therapy for advanced non-small cell lung cancer. AREAS COVERED: This review analyzes the biology of the target antigen, summarizes preclinical studies and discusses clinical trials in adults and the pediatric experience with racotumomab. EXPERT OPINION: Proper patient selection and combination with chemotherapy, radiotherapy or checkpoint inhibitors appear to be critical issues to maximize the effects of racotumomab vaccination in lung cancer. In a recent phase I clinical trial in children with relapsed or resistant neuroectodermal malignancies, racotumomab was well tolerated and immunogenic, and its evaluation as immunotherapy for high-risk neuroblastoma is warranted.

A Phase I Study of the Anti-Idiotype Vaccine Racotumomab in Neuroblastoma and Other Pediatric Refractory Malignancies.

BACKGROUND: Pediatric neuroectodermal malignancies express N-glycolylated gangliosides including N-glycolyl GM3 (NeuGcGM3) as targets for immunotherapy. PROCEDURE: We evaluated the toxicity and maximum tolerated dose and immunological response of racotumomab, an anti-idiotype vaccine targeting NeuGcGM3 through a Phase I study enrolling children with relapsed or resistant tumors expressing NeuGcGM3. MATERIALS AND METHODS: Drug dose was escalated to three levels (0.15-0.25-0.4 mg) of racotumomab administered intradermally. Each drug level included three patients receiving a total of three doses, every 14 days. A confirmation cohort was added to the highest dose level. Antibody response was assessed upon study entry and at 4-week intervals for at least three immunological determinations for each patient. RESULTS: Fourteen patients were enrolled (10 with neuroblastoma, one with retinoblastoma, one with Wilms' tumor, and two with brainstem glioma). Three patients completed the three drug levels and three were enrolled in the confirmation cohort. One patient died of tumor progression before completing the three applications. Racotumomab was well tolerated. The only side effect observed was grade 1-2 toxicity at the injection site. Racotumomab elicited an IgM and/or IgG antibody response directed against NGcGM3 in nine patients and IgM against racotumomab in 11 of 13 evaluable patients. The maximum tolerated dose was not reached and no dose-limiting toxicity was seen. CONCLUSIONS: Racotumomab vaccination has a favorable toxicity profile up to a dose of 0.4 mg, and most patients elicited an immune response. Its activity as immunotherapy for neuroectodermal malignancies will be tested in further clinical trials.

Association of Cone-Rod Homeobox Transcription Factor Messenger RNA With Pediatric Metastatic Retinoblastoma.

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.

Preclinical evaluation of racotumomab, an anti-idiotype monoclonal antibody to N-glycolyl-containing gangliosides, with or without chemotherapy in a mouse model of non-small cell lung cancer.

N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule usually found in mammalian cells as terminal constituents of different membrane glycoconjugates such as gangliosides. The NeuGcGM3 ganglioside has been described as a tumor antigen for non-small cell lung cancer (NSCLC) in humans. Racotumomab is an anti-NeuGc-containing gangliosides anti-idiotype monoclonal antibody (mAb) (formerly known as 1E10) that has received attention as a potential active immunotherapy for advanced lung cancer in clinical trials. In this work, we have examined the antitumor activity of racotumomab in combination or not with chemotherapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Vaccination with biweekly doses of racotumomab at 50-200 µg/dose formulated in aluminum hydroxide (racotumomab-alum vaccine) demonstrated a significant antitumor effect against the progression of lung tumor nodules. Racotumomab-alum vaccination exerted a comparable effect on lung disease to that of pemetrexed-based chemotherapy (100 mg/kg weekly). Interestingly, chemo-immunotherapy was highly effective against lung nodules and well-tolerated, although no significant synergistic effect was observed as compared to each treatment alone in the present model. We also obtained evidence on the role of the exogenous incorporation of NeuGc in the metastatic potential of 3LL cells. Our preclinical data provide support for the combination of chemotherapy with the anti-idiotype mAb racotumomab, and also reinforce the biological significance of NeuGc in lung cancer.

Racotumomab: an anti-idiotype vaccine related to N-glycolyl-containing gangliosides - preclinical and clinical data.

Neu-glycolyl (NeuGc)-containing gangliosides are attractive targets for immunotherapy with anti-idiotype mAbs, because these glycolipids are not normal components of the cytoplasmic membrane in humans, but their expression has been demonstrated in several human malignant tumors. Racotumomab is an anti-idiotype mAb specific to P3 mAb, an antibody which reacts to NeuGc-containing gangliosides, sulfatides, and other antigens expressed in tumors. Preparations containing racotumomab were able to induce a strong anti-metastatic effect in tumor-bearing mice. Different Phase I clinical trials have been conducted in patients with advanced melanoma, breast cancer, and lung cancer. The results of these clinical trials demonstrated the low toxicity and the high immunogenicity of this vaccine. The induced antibodies recognized and directly killed tumor cells expressing NeuGcGM3. A Phase II/III multicenter, controlled, randomized, double blind clinical trial was conducted to evaluate the effect of aluminum hydroxide-precipitated racotumomab vaccine in overall survival in patients with advanced non-small cell lung cancer. The clinical results of this study showed a significant clinical benefit in the patients who were treated with the anti-idiotype vaccine.

Antitumor protection by NGcGM3/VSSP vaccine against transfected B16 mouse melanoma cells overexpressing N-glycolylated gangliosides.

BACKGROUND: Cancer vaccines are designed to modulate immunological responses against tumor cells through the presentation of tumor antigens. MATERIALS AND METHODS: The mouse mRNA of the cytidine monophospho-N-acetylneuraminic acid hydroxylase (Cmah) gene, the enzyme that catalyzes the synthesis of N-glycolylneuraminic acid (NGc), was cloned and transfected into the B16 melanoma cell line. Transfected cells (B16-H) were characterized and used as an NGcGM3-positive primary tumor model for the evaluation of the therapeutic activity of the NGcGM3/VSSP vaccine. RESULTS: The presence of NGcGM3 in B16-H cells promoted proliferation and adhesion in vitro, but resulted in reduced tumorigenicity in vivo. However, B16-H cells developed growing tumors in mice where NGcGM3/VSSP vaccination induced a therapeutic antitumor activity. NGcGM3/VSSP was ineffective in mice inoculated with parental B16 or B16-H cells that had lost NGcGM3 expression. CONCLUSION: The presence of NGcGM3 in tumor cells is critical for the antitumor activity of NGcGM3/VSSP vaccine.

Detection of N-glycolyl GM3 ganglioside in neuroectodermal tumors by immunohistochemistry: an attractive vaccine target for aggressive pediatric cancer.

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 µm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.

NGcGM3 ganglioside: a privileged target for cancer vaccines.

Active specific immunotherapy is a promising field in cancer research. N-glycolyl (NGc) gangliosides, and particularly NGcGM3, have received attention as a privileged target for cancer therapy. Many clinical trials have been performed with the anti-NGc-containing gangliosides anti-idiotype monoclonal antibody racotumomab (formerly known as 1E10) and the conjugated NGcGM3/VSSP vaccine for immunotherapy of melanoma, breast, and lung cancer. The present paper examines the role of NGc-gangliosides in tumor biology as well as the available preclinical and clinical data on these vaccine products. A brief discussion on the relevance of prioritization of cancer antigens in vaccine development is also included.

Direct validation of NGcGM3 ganglioside as a new target for cancer immunotherapy.

OBJECTIVE: The target concept means not only an aberrant expression of a particular molecule in tumour tissues but also evidence of a clear therapeutic advantage, as a consequence of immune-intervention, in an antigen-positive relevant tumour model. Since we reported the presence of NGcGM3 ganglioside in human breast tumours years ago and though Phase I clinical trials of a ganglioside containing vaccine have been conducted, a definitive direct validation of this peculiar molecule as target for cancer immunotherapy has remained unperformed. METHODS: Two animal models were used: leghorn chickens and C57BL/6 mice. The murine 3LL-D122 cell line, the derived subcutaneous tumours and metastatic lung lesions were processed for gangliosides identification. Active immunotherapy experiments in the 3LL-D122 spontaneous lung metastasis model were performed with NGcGM3/VSSP vaccine prepared by conjugation of NGcGM3 with the outer membrane proteins of Neisseria meningitides. RESULTS: The 3LL-D122 Lewis lung carcinoma results were consistent with an increased expression of NGcGM3 from primary tumours to metastatic lesions, as observed in human breast cancer samples. Both vaccines, prepared with synthetic or natural-source-derived ganglioside, showed similar anti-tumour and immunogenicity profiles. Finally, a clear involvement of NK1.1(+) cells and CD8(+) T cells in the anti-metastatic effect elicited by the vaccine was manifested. CONCLUSIONS: While 'proof of concept' Phase II and III clinical trials with the NGcGM3/VSSP vaccine in cancer patients are currently ongoing these results reasonably sustain the validation of this peculiar ganglioside as a novel target for cancer immunotherapy.

Optimization of molecular detection of GD2 synthase mRNA in retinoblastoma.

Extraocular dissemination is the main cause of death in patients with retinoblastoma (RB) in developing countries, and there are few molecular markers that are useful for the evaluation of minimal disseminated disease. The GD2 ganglioside is known to be expressed by RB cells that metastasize in bone marrow, and the activity of the enzyme responsible for its synthesis, GD2 synthase, can be detected in neuroblastoma, which shares many phenotypic features with RB. The purpose of the present study was to optimize the detection of GD2 synthase expression by reverse transcription-polymerase chain reaction (RT-PCR) followed by nested-PCR in human RB cell lines and patient samples. The optimization strategy was carried out using the RB cell lines Y79 and WERI-Rb1 and specific primers designed for the human sequence of GD2 synthase mRNA. We detected GD2 synthase expression with at least 200 and 40 pg of total RNA extracted from cultured RB cells using a first round of RT-PCR amplification or a second round of nested-PCR, respectively. We also confirmed the expression of GD2 synthase by RT-PCR and immunohistochemical detection of the ganglioside in human RB tumors xenotransplanted in nude mice. Using tumor bank specimens from eight RB patients, we were able to demonstrate the presence of GD2 synthase mRNA in blood and cerebrospinal fluid samples in cases of extraocular dissemination of the tumor. The sequence was not detected in samples derived from children with low-risk disease or healthy adult volunteers. Hence, GD2 synthase mRNA detection through an optimized nested RT-PCR assay is a promising tool for the assessment of minimal disseminated disease in enucleated patients.

Detection and characterization of N-glycolyated gangliosides in Wilms tumor by immunohistochemistry.

Gangliosides are glycolipids present on the cell surface. The N-glycolylated ganglioside NeuGc-GM3 has been described in some neoplasms, such as breast carcinoma and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in Wilms tumor by immunohistochemistry. Postchemotherapy tumors were grouped into different histologic subtypes considering the main preserved component. Formalin-fixed, paraffin-embedded tumor samples were cut into 5-microm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Sections from breast carcinoma were employed as positive controls. Presence of NeuGc-GM3 was evident in 22 of 25 (88%) cases. The staining was stronger in the epithelial component, with a membrane pattern and cytoplasmic diffusion. The stromal component expressed cytoplasmic NeuGc-GM3 in cells with rhabdomyoblastic differentiation. Tubules of adjacent renal tissue were also positive, but no expression of NeuGc-GM3 was detected in nontumoral fetal kidney. Until now, the expression of N-glycolylated gangliosides in pediatric solid tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of Wilms tumors, suggesting its potential utility as a specific target of immunotherapy.

Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines.

BACKGROUND: Carbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc) is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues. METHODS: We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth. RESULTS: In the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone. CONCLUSION: Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.

[Molecular detection of minimal residual disease in melanoma and solid tumors]. / Detección molecular de enfermedad mínima residual en melanoma y otros tumores sólidos.

The availability of highly sensitive and specific methods for the detection of minimal residual disease in patients with solid tumors may have important prognostic and therapeutic implications. One of the most widely used methods for the molecular detection of cancer cells is the RT-PCR technique, which leads to the amplification of tissue-specific mRNA. It was firstly applied in the detection of circulating tumor cells in peripheral blood of patients with advanced melanoma; and soon it was adapted for the detection of minimal residual disease in other solid tumors. The aim of the present review is to evaluate the published data since the first study in 1991 and to analyze the clinical value of the findings obtained. The importance of sample handling and standardization of RT-PCR procedures is also discussed.

Detección molecular de enfermedad mínima residual en melanoma y otros tumores sólidos / Molecular detection of minimal residual disease in melanoma and solid tumors

La disponibilidad de métodos altamente sensibles y específicos para la detección de enfermedad mínima residual en pacientes con tumores sólidos podría tener importantes consecuencias pronósticas y terapéuticas. Uno de los métodos más usados para la detección molecular de células cancerosas es la técnica de RT-PCR, que permite la amplificación de secuencias de ARNm específicas de distintos tejidos. La misma fue aplicada por primera vez en la detección de células tumorales circulantes en sangre periférica de pacientes con melanoma avanzado, poco tiempo después fue adaptada para la búsqueda de enfermedad mínima residual en otros tumores sólidos. El objetivo de la presente revisión es evaluar la información publicada desde el primer estudio sobre este tema en 1991 y analizar el valor clínico de los hallazgos obtenidos. Se discute también la importancia del manejo de la muestra y de la estandarización de los procedimientos de RT-PCR.

The availability of highly sensitive and specific methods for the detection of minimal residual disease in patients with solid tumors may have important prognostic and therapeutic implications. One of the most widely used methods for the molecular detection of cancer cells is the RT-PCR technique, which leads to the amplification of tissue-specific mRNA. It was firstly applied in the detection of circulating tumor cells in peripheral blood of patients with advanced melanoma; and soon it was adapted for the detection of minimal residual disease in other solid tumors. The aim of the present review is to evaluate the published data since the first study in 1991 and to analyze the clinical value of the findings obtained. The importance of sample handling and standardization of RT-PCR procedures is also discussed.