Mepur H. Ravindranath

Prolonged survival of patients receiving active immunotherapy with Canvaxin therapeutic polyvalent vaccine after complete resection of melanoma metastatic to regional lymph nodes.

ObjectiveTo determine whether adjuvant postoperative active specific immunotherapy with a therapeutic polyvalent vaccine (PV) called Canvaxin can prolong survival following complete resection of melanoma metastatic to regional nodes (American Joint Committee on Cancer [AJCC] stage III melanoma). Summary Background DataDespite complete lymphadenectomy, 5-year overall survival (OS) for patients with melanoma metastatic to regional lymph nodes is only 20% to 50%, depending on the number of tumor-involved nodes. In 1984, the authors began phase II trials of Canvaxin PV as postsurgical adjuvant therapy for AJCC stage III melanoma. MethodsPatients who received PV between 1984 and 1998 were compared with patients who did not receive PV postsurgical therapy between 1971 and 1998. The seven covariates recently defined by the AJCC Melanoma Staging Committee (number of metastatic nodes, palpable status, ulceration, age, primary site, pT stage, and gender) were included by Cox regression in a multivariate model of OS. A computerized program matched PV and non-PV patients by these covariates. ResultsOf 2,602 patients who underwent complete lymphadenectomy for AJCC stage III melanoma with regional nodal metastases and were followed up by the same team of oncologists between 1971 and 1998, 935 received PV and 1,667 did not. Median OS and 5-year OS were significantly higher in PV than non-PV patients (56.4 vs. 31.9 months and 49% vs. 37%, respectively;P = .0001). When the non-PV patients were matched by the four most significant covariates, 447 matched pairs were formed between patients seen before or after January 1, 1985, and the OS was not different between the two time periods (P = .789). However, when the PV patients were matched with non-PV patients by six covariates forming 739 pairs, the PV patients survived longer (P = .0001). Detailed analysis of the 1,505 patients who were seen or who began vaccine therapy within 4 months after lymphadenectomy, and who had more complete data on the seven prognostic covariates showed that median OS and 5-year OS were higher in 445 PV patients than in 1,060 non-PV patients: 70.4 versus 31 months and 52% versus 37%, respectively (P = .0001). Multivariate Cox regression analysis identified six significant prognostic factors: number of metastatic nodes, size of metastatic nodes, pT stage, ulceration, age, and PV therapy. PV therapy reduced the relative risk of death to 0.64 (95% confidence interval, 0.55–0.76) (P = .0001); sex and site of primary were of borderline significance. ConclusionsThis large single-institution study independently confirmed the significance of prognostic covariates in the new AJCC staging system. By using modern statistical methods that controlled for all known prognostic factors, it also demonstrated PV’s ability to significantly enhance OS. A multicenter phase III randomized trial is underway to validate the efficacy of PV as a postsurgical adjuvant.

Ganglioside GM3:GD3 ratio as an index for the management of melanoma.

This report evaluates the relevance of the ratio of the melanoma‐associated ganglioside, GD3, and its precursor, GM3, to prognosis and management of Stage II disease. Tumor biopsy specimens from 42 melanoma patients were examined for the ratio and found that GD3 and GM3 constitute 80% of the total lipid bound sialic acids (LBSA) of melanoma. Although the ratio of GM3:GD3 in melanocytes, the progenitors of melanoma, is 19:1 (based on %LBSA), it ranged from 15:1 to 1:5 in tumor biopsy specimens. Patients are categorized into three groups based on their GM3:GD3 ratio (%LBSA) of tumor tissues as Group I (ratio ranged from 15:1 to 1.5:1) (10 of 42), Group II (1.4:1 to 1:1.4) (13 of 42), and Group III (1:1.5 to 1:5) (19 of 42). When the overall survival of patients from the onset of Stage II disease was evaluated among different groups, patients belonging to Group I survived significantly longer than patients of Group II (P = 0.02) and Group III (P = 0.01). Interestingly, Group III expressed immunogenic gangliosides (GD2, GM2, and O‐AcGD3) better than the other groups and may benefit more from therapies targeted against these gangliosides antigens. The results of this study indicate that GM3:GD3 ratio is an unusual but well‐defined biochemical criteria that may be useful for prognosis and therapeutic management of the disease. Therefore, we propose a routine analysis of the GM3:GD3 ratio of tumors excised after surgery. Screening the ratio is feasible since melanoma expresses a simple profile of gangliosides unlike other forms of cancer.

Epicatechins Purified from Green Tea (Camellia sinensis) Differentially Suppress Growth of Gender-Dependent Human Cancer Cell Lines

The anticancer potential of catechins derived from green tea is not well understood, in part because catechin-related growth suppression and/or apoptosis appears to vary with the type and stage of malignancy as well as with the type of catechin. This in vitro study examined the biological effects of epicatechin (EC), epigallocatechin (EGC), EC 3-gallate (ECG) and EGC 3-gallate (EGCG) in cell lines from human gender-specific cancers. Cell lines developed from organ-confined (HH870) and metastatic (DU145) prostate cancer, and from moderately (HH450) and poorly differentiated (HH639) epithelial ovarian cancer were grown with or without EC, EGC, ECG or EGCG. When untreated cells reached confluency, viability and doubling time were measured for treated and untreated cells. Whereas EC treatment reduced proliferation of HH639 cells by 50%, EGCG suppressed proliferation of all cell lines by 50%. ECG was even more potent: it inhibited DU145, HH870, HH450 and HH639 cells at concentrations of 24, 27, 29 and 30 µM, whereas EGCG inhibited DU145, HH870, HH450 and HH639 cells at concentrations 89, 45, 62 and 42 µM. When compared with EGCG, ECG more effectively suppresses the growth of prostate cancer and epithelial ovarian cancer cell lines derived from tumors of patients with different stages of disease.

Differential Growth Suppression of Human Melanoma Cells by Tea (Camellia sinensis) Epicatechins (ECG, EGC and EGCG)

We previously reported that catechins of green tea have different antiproliferative effects on cell lines derived from gender-dependent cancers; epicatechin 3-gallate (ECG) had the strongest inhibitory effect. In the present study, we examined the effects of epigallocatechin (EGC), epicatechin-gallate (ECG) and EGC 3-gallate (EGCG) on the viability, density, doubling time and cycle number of cell lines derived from melanoma metastasized to lymph nodes (MB-1133 and SE-0154) or distant organs (CH-0356, JK-0346, SA-1171, GE-0208, NS-1176 and LF-0023). These catechins have been documented to have no growth suppressive or apoptotic effects on normal melanocytes (Nihal et al., Int J Cancer 2005;114:513–21). EGCG (50 μM) showed greater inhibitory potency than EGC (50 μM) in SE-0154, NS-1176, GE-0208 and LF-0023 cell lines but the two catechins produced similar inhibitory effects in CH-0356, JK-0346 and SA-1171 cell lines. The IC50 (50% inhibitory concentration) was lower for EGC than EGCG in MB-1133 and CH-0356 cells, higher for EGC than EGCG in GE-0208 cells and comparable (11–12 μM) for both the catechins in LF-0023 cells. When compared with EGC, the cytotoxic effect (% dead cell counts) and the suppression of the growth (change in cell number) of all melanoma cell lines tested were pronounced with EGCG. This investigation validates the hypothesis that anticancer action of the various catechins may vary with the type of malignancy and provides a model for tumor cell heterogeneity based on susceptibility and resistance of tumor cells to different green tea catechins. Therefore, this information is critical for undertaking chemopreventive or chemotherapeutic trials against melanoma and gender-based cancers.

Chapter 19 Tumor gangliosides as targets for active specific immunotherapy of melanoma in man

Publisher Summary This chapter discusses various important aspects of gangliosides as vaccines for active specific immunotherapy of melanoma. The chapter presents new data on the antibody response to gangliosides induced in patients receiving Merkel cell polyomavirus. Several lines of evidence indicate that gangliosides modulate immune functions. Among the documented immunoregulatory properties of gangliosides are (1) inhibition of normal human lymphoproliferative responses to mitogens and antigens, (2) inhibition of interleukin-2 dependent cell proliferation, (3) inhibition of cytotoxic effector functions, and (4) binding to helper cells and modulated expression of CD4. Gangliosides have also been shown to modulate the humoral immune response. Melanoma cells in the radial growth phase have a GM3 and GD3 profile similar to that of normal melanocytes. GM3 and GD3 inhibits the proliferation of human lymphocytes induced by phytohemagglutinin (PHA) and interleuhn-2. Further, studies show that gangliosides—particularly GM3—significantly inhibits the concanavalin A-induced proliferation of peripheral blood lymphocytes. The lymphocytes of melanoma patients seemed to be more sensitive to the influence of exogenous gangliosides than did those of healthy controls. The same was true for mixed lymphocyte reactions.

Endothelial‐selectin ligands sialyl Lewisx and sialyl Lewisa are differentiation antigens immunogenic in human melanoma

Sialyl Lewisx (sLex) and sialyl Lewisa (sLea), the endothelial‐selectin ligands involved in extravasation of neutrophils and carcinomas, have been identified in human melanoma. This study explored the following issue: If these ligands are immunogenic tumor‐differentiation antigens, they would be potential targets for immunotherapy because of their putative roles in extravasation and metastasis.

Role of Gangliosides in Active Immunotherapy with Melanoma Vaccine

Among various tumor associated cell surface antigens, gangliosides, the glycosphingolipids that contain sialic acids, offer a variety of epitopes, some of which are preferentially expressed on melanoma cells. These surface components of the bilayered lipid membrane of tumor cells are the targets of active immunotherapy with melanoma vaccine. Purified gangliosides in aqueous solution form micelles and, at high density, form lactones. Their antigenic expression (physical conformation and orientation) on the cell surface is governed by the nature of the sphingosine and the fatty acids they contain. Evidence is accruing to show that the nature of the fatty acid moiety of gangliosides differs in normal and neoplastic cells. Gangliosides per se are not immunogenic and require extrinsic adjuvanticity. Preparation of a melanoma cell vaccine for active immunotherapy requires an understanding of the ganglioside profile of melanoma, the ganglioside-associated heterogeneity of melanoma, and the role of shed melanoma gangliosides in the immunosuppression of cell mediated and humoral immunity. In addition, the role of some of the anti-ganglioside antibodies in the elimination of shed gangliosides, the cytotoxic killing of tumor cells, as well as in the down-regulation of lymphocyte functions must be considered in the formulation of vaccine. Different strategies for augmenting the immunogenicity of melanoma associated gangliosides with melanoma vaccine are evaluated.

Cryosurgical ablation of liver tumors in colon cancer patients increases the serum total ganglioside level and then selectively augments antiganglioside IgM

Cryosurgical ablation (CSA) of tumors induces disruptive necrosis. Necrosis may release tumor gangliosides into circulation and they may augment serum antiganglioside antibodies depending on the nature of gangliosides released. The hypothesis is tested by determining the level of serum total gangliosides (STG) and their antibody titers in the sera of colon cancer patients with cryoablated liver tumors. As controls, we examined the sera of patients who underwent radiofrequency ablation (RFA) and regular surgery (RS), none of which cause disruptive necrosis. The STG level (expressed as lipid-bound sialic acids, LBSA) is higher (p(2)<0.001) in 35 patients (stage IV) than in 38 healthy case-controls (median 23.48 mg/dL, Q-range 7.1 vs 16.04 mg/dL, Q-range 4.5). The mean STG level increased significantly to 31.2+/-6.0mg/dL (p(2)<0.03) after CSA. Concomitantly, the IgM titer against colon cancer-associated gangliosides (GM(2), GD(1a), GT(1b)), increased significantly, but no increase was observed against normal tissue gangliosides (GM(3) or GM(1)). Also after RFA and RS, no such increase was observed either in the level of STG or in IgM titer against tumor gangliosides. The results suggest that CSA-induced necrosis might have acted as an adjuvant, because purified gangliosides without exogenous adjuvants even after repeated immunization failed to elicit antibody response. The post-CSA decline in the STG level correlated with the increase in the antibodies, suggesting a homeostatic role of the antibodies.

Endogenous immune response to gangliosides in patients with confined prostate cancer

Our study investigated whether endogenous IgM antibodies to gangliosides occur in patients with early stages of prostate cancer (CaP) patients, after defining ganglioside profiles of CaP cell lines. Immune and resorcinol staining detected the presence of gangliosides GM3, GM2, GD3, GD2 and GD1a but not GM1a, GD1b or GT1b in the extracts of normal prostatic epithelial cells (PrEC) and neoplastic androgen‐insensitive (PC‐3, DU145) and ‐sensitive (LNCaP‐FGC and LNCaP‐FGC‐10) CaP cells. Using a sensitive ELISA, developed and validated in our laboratory, the titers of IgM against 8 gangliosides from sera of patients with benign prostatic hyperplasia (BPH) (n = 11), organ‐confined (T1/T2, n = 36) and unconfined (T3/T4, n = 27) CaP and age‐matched healthy men (n = 11) were determined double‐blinded. Using ANOVA and Fishers least significant difference (LSD) methods, the log‐titers among different groups were compared. CaP patients differed from healthy and BPH patients in increased titers against GD1a and decreased titers against GD3. Titers of antibodies to other gangliosides exhibited no difference between CaP patients and others. The specific augmentation of anti‐GD1a IgM in patients with organ‐confined CaP (stage T1/T2) but not in patients with unconfined CaP (stage T3/T4) or BPH or in healthy controls is striking. This finding together with identification of GD1a as a major ganglioside in CaP cell lines and with the accruing studies on the immunosuppressive nature of GD1a indicates that augmentation of anti‐GD1a IgM in confined CaP may signify an early endogenous immune response to eliminate a “danger signal” from tumor microenvironment and circulation.

Serum Anti-ganglioside Igm Antibodies in Soft Tissue Sarcoma: Clinical Prognostic Implications

PURPOSEGangliosides are tumor-associated antigens with many biologic functions, including complex interactions with cytokines and other modulators of the immune system. Serum total ganglioside level may be an ideal surrogate marker to predict tumor burden and response to treatment. Antibodies produced against tumor gangliosides may help predict survival. The purpose of this study is to determine whether the serum total ganglioside levels might predict the tumor burden in patients with soft tissue sarcoma, and whether the augmented anti-ganglioside immunoglobulin M (IgM) response might reflect the clinical outcome of these patients. METHODSSerum TG levels were measured in the cryopreserved sera by estimating lipid-associated sialic acids from 97 patients before surgical resection of soft tissue sarcoma and from 39 age- and gender-matched healthy volunteers. All sera were analyzed for IgM titers (expressed natural log) by enzyme-linked immunosor-bent assay against eight gangliosides (GM1, GM2, GM3, GD3, GD2, GD1a, GD1b, and GT1b). Cox regression was used for univariate and multivariate analyses of the variables affecting progression-free and overall survival. RESULTSSerum TG levels were higher in soft tissue sarcoma patients than in healthy individuals (21.8 + 7.7 vs 16.1 + 2.7 mg/dL; P = 0.001). Larger tumors, high histologic grade, and more advanced stage of disease correlated with higher serum total ganglioside levels (P ≤0.05). Anti-ganglioside titers to GM3, GD2, and GT1b were significantly higher in patients with soft tissue sarcoma, whereas anti-GD1a and GD1b titers were significantly higher in healthy subjects. The titers of antibodies against GM1, GM2, and GD3 in patients with soft tissue sarcoma were comparable to those of the healthy individuals. When compared with healthy controls, patients with low-grade tumors had higher titers of anti-GT1b, anti-GM3, and anti-GD2 antibodies, and patients with high-grade tumors had higher titers of anti-GT1b and anti-GD2 antibodies. These data suggest that the predominant ganglio-sides expressed by sarcomas may include GT1b and GD2. In addition, low-grade tumors may express an immunogenic species of GM3. On both univariate and multivariate analyses, augmented anti-GD1a IgM titers, age > 50 years, and retroperitoneal location were predictive of decreased overall survival, whereas augmented anti-GT1b titers were predictive of improved overall survival. CONCLUSIONSSerum TG level may be a useful marker of tumor burden and response to treatment for soft tissue sarcoma. Anti-GD1a and anti-GT1b IgM titers predicted survival and may be of therapeutic and prognostic value in the management of soft tissue sarcoma.