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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 Fisher's 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.

Human melanoma cells (from biopsies and culture) express sialyl-Lewis x and sialyl Lewis a , the ligands for ECAM. These ligands may facilitate tumor progression and metastasis in human cancers. To test whether the antibodies to these ligands inhibit tumor progression, IgG and IgM responses to sLe x and sLe a were induced in C57BL/6j mice (n = 76) by immunization with human melanoma cells, with or without adjuvants (BCG, MPL, KLH). Control mice were treated with saline or BCG. Tumor growth and antigen expression were monitored after challenge with B16 mouse melanoma cells expressing sLe x , sLe a and the ganglioside GM 3 . Tumor growth was reduced in mice immunized with BCG alone or cells with BCG or MPL, while tumors in mice receiving cells without adjuvants grew larger than in the control. Augmentation of IgM titers to sLe x and Gl4 3 after immunization with BCG, or with cells with BCG or MPL correlated with retarded tumor growth, while increased IgG titers to sLe x significantly correlated with aggressive tumor growth in mice immunized with cells without adjuvants. SLe x , sLe a and GM 3 were expressed in tumors from mice treated with saline or BCG. SLe x expression, in particular, was lost in tumors growing in mice immunized with cells with or without adjuvants. Anti-sLe x antibodies may promote or prevent tumor growth by antigenic modulation or by cytotoxic killing of tumor cells. Since early anti-sLe x IgM correlated with tumor regression, in contrast to anti-sLe x IgG, it may serve as a potential early endpoint for the effectiveness of melanoma vaccines expressing the antigens. Int.

Phenotypic expression of human leukocyte antigen (HLA)-E on the surface of tumor lesions includes intact heterodimer [HLA-E heavy chain and β2-microglobulin (β2m)] and β2m-free monomer. Anti-HLA-E monoclonal antibodies (mAbs), MEM-E/02 or 3D12 bind to the peptide sequences in β2m-free HLA-E, which is common and shared with HLA-Ia monomers. A newly developed monospecific anti-HLA-E mAb (TFL-033) recognizes HLA-E-restricted peptide sequences on α1 and α2 helices away from β2-m-site. Tumor progression may involve shedding of β2-m from HLA-E or overexpression of β2m-free monomers. There is a need to identify and distinguish the different phenotypic expression of HLA-E, particularly the intact heterodimer from the β2m-free monomer on the surface of tumor lesions. Because of the unique peptide-binding affinities of the mAbs, it is hypothesized that TFL-033 and MEM-E/02 may distinguish the phenotypic expressions of cell surface HLA-E during stages of tumor progression. Only TFL-033 stained diffusely the cytoplasm of normal mucosa. The incidence and intensity of TFL-033 staining of the cell surface in early stages, poorly or undifferentiated and non-nodal lesions and in diffuse carcinoma is greater than that of MEM-E/02. Whereas MEM-E/02 stained terminal stages, adenocarcinoma and lymph node metastatic lesions intensely, either owing to increased expression of β2m-free HLA-E with tumor progression or owing to expression of HLA-Ia molecules. Our study evaluates the relative diagnostic potential of HLA-E-monospecific TFL-033 and the HLA-Ia-reactive MEM-E/02 for determining the specific distribution and immunodiagnosis of different phenotypic expression HLA-E in tumor lesions, and the structural and functional alterations undergone by HLA-E during tumor progression.

A mechanistic marker correlating with tumor progression and clinical response is useful for assessing therapeutic response and determining the course of therapy. Since serum-total-ganglioside (sTG) and antiganglioside-IgM antibody levels reflected tumor progression, the feasibility of utilizing sTG for assessing the response to immunotherapy of metastatic-melanoma was tested. Patients (n = 34) were immunized with dendritic cells cocultured with irradiated, IFN gamma-treated autologous tumor cells admixed with GM-CSF. Levels of sTG and antiganglioside-IgM antibody titers were measured in sera of vaccine recipients at 0, 4 and 24 weeks of treatment. Based on sTG-level, whether lower (L) or higher (H) than the mean + 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts-I (LLL, n = 16), II (HHL/HLL, n = 4), III (LLH/LHH/LHL, n = 7) and IV (HHH/HLH, n = 7). The cohorts were regrouped as sTG- downregulators (sTG-DR; n = 20) and upregulators (sTG-UR; n = 14). These two cohorts differed significantly in their overall (p0.012) and progression-free (p = 0.0001) survival post-treatment. 43% sTG-UR died within 39 months, with a median survival of 39 months, whereas 61% of the sTG-DR survived for 48 months. Both endogenous and vaccine-induced antiganglioside-IgM antibodies appeared to regulate sTG levels. Nonresponders had increased sTG with no or low IgM antibody response. The sTG level is regulated within 24 weeks post-treatment and therefore, may serve as an ideal biomarker for assessing therapeutic responses in patients. Clinical correlations of sTG indicate that sTG-downregulating therapy may be an effective treatment strategy for melanoma.