High Antigen Expression Research Articles

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines

BackgroundDespite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines.ResultTo compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited.ConclusionThrough systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus insoluble forms of the protein antigens. If an antigen, such as EGFP, is soluble and expressed at high levels, a low-copy plasmid-cytoplasmic expression strategy is recommended; since it provokes the highest B cell responses and also induces good T cell responses. If a T cell response is preferred, a eukaryotic expression plasmid or a chromosome-based, cytoplasmic-expression strategy is more effective. For insoluble antigens such as HA, an outer membrane expression strategy is recommended.

High expression of prostate-specific membrane antigen in the tumor-associated neo-vasculature is associated with worse prognosis in squamous cell carcinoma of the oral cavity

AbstractProstate-specific membrane antigen (PSMA) is a transmembrane protein expressed in prostate cancer as well as in the neo-vasculature of nonprostatic solid tumors. Here, we determined the expression pattern of PSMA in the vasculature of oral squamous cell carcinoma. Using a previously validated antibody, PSMA staining distribution and cyclooxygenase 2 (COX2) expression status was evaluated in a cohort of patients with squamous cell carcinoma of the oral cavity (n=96) using immunohistochemistry and was correlated with clinicopathological features as well as outcome. Twenty-four (25%) cases showed no detectable PSMA staining, 48 (50%) demonstrated positive immunoreactivity for PSMA in less than 50% of microvessels and 24 (25%) cases showed strong endothelial PSMA expression in more than 50% of tumor-associated microvessels. High endothelial PSMA expression was associated with greatly reduced survival (18.2 vs 77.3 months; P=0.0001) and maintained prognostic significance after adjusting for grade and stage in multivariate analysis (hazard ratio=2.19, P=0.007). Furthermore, we observed a strong association between endothelial PSMA and cancer cell-specific COX2 expression. In conclusion, we provide the first evidence for the prognostic significance of endothelial PSMA expression in oral squamous cell carcinoma and, suggest a potential interaction between arachidonic acid metabolites and endothelial PSMA expression in the tumor neo-vasculature.

Luminal A and luminal B (HER2 negative) subtypes of breast cancer consist of a mixture of tumors with different genotype

BackgroundThe St Gallen International Expert Consensus 2011 has proposed a new classification system for breast cancer. The purpose of this study was to elucidate the relationship between the breast cancer subtypes determined by the new classification system and genomic characteristics.MethodsInvasive breast cancers (n = 363) were immunohistochemically classified as follows: 111 (30.6%) as luminal A, 95 (26.2%) as luminal B (HER2 negative), 69 (19.0%) as luminal B (HER2 positive), 41 (11.3%) as HER2, and 47 (12.9%) as basal-like subtypes.ResultsThe high expression of Ki-67 antigen was detected in 236 tumors; no cases of luminal A subtype showed high expression of the Ki-67 antigen, but more than 85% of tumors of the other subtypes showed high expression. In addition, DNA ploidy and chromosomal instability (CIN) were assessed using imaging cytometry and FISH, respectively. In this series, 336 (92.6%) tumors consisted of 129 diploid/CIN- and 207 aneuploid/CIN + tumors. Diploid/CIN- and aneuploid/CIN+ features were detected in 64.9% and 27.9% of luminal A, 41.1% and 49.5% of luminal B (HER2-), 11.6% and 81.2% of luminal B (HER2+), 4.9% and 90.2% of HER2, and 17.0% and 76.6% of basal-like subtypes, respectively. Unlike the luminal B (HER2+), HER2 and basal-like subtypes, the luminal A and luminal B (HER2-) subtypes were heterogeneous in terms of DNA ploidy and CIN.ConclusionsIt is reasonable to propose that the luminal A and luminal B (HER2-) subtypes should be further divided into two subgroups, diploid/CIN- and aneuploid/CIN+, based on their underlying genomic status.

Pretreatment of leukemic cells with low-dose decitabine markedly enhances the cytotoxicity of gemtuzumab ozogamicin

Prognosis in relapsed/refractory acute myeloid leukemia (AML) is poor due to drug resistance. Gemtuzumab ozogamicin (GO) is a molecularly targeted drug, which consists of anti-CD33 antibody and calicheamicin. The high expression of CD33 antigen on most AML cells and exceptionally marked cytotoxicity of calicheamicin have attracted strong interest in GO, but clinical results on GO monotherapy have been disappointing, and methods of overcoming resistance to GO have accordingly been sought.

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8(+) T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.

Erratum to: High expression of MAGE-A10 cancer-testis antigen in triple-negative breast cancer

Erratum to: High expression of MAGE-A10 cancer-testis antigen in triple-negative breast cancer

High expression of MAGE-A10 cancer-testis antigen in triple-negative breast cancer

Recent studies indicate that ER/PR/HER-2-negative (triple-negative, TN) breast cancers may be "CTA-rich" tumors, suggesting the possibility of CTA-based cancer vaccines as a treatment option for patients bearing these tumors. MAGE-A10 together with NY-ESO-1 is probably the most immunogenic CTA, representing a potentially highly attractive target of active specific immunotherapies. Paraffin-embedded tumor sections were collected retrospectively from 165 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. The expression of MAGE-A10 and NY-ESO-1 was correlated with other clinicopathological variables. MAGE-A10 expression (score ≥ 2+) was detected in 105/164 (64%), and NY-ESO-1 expression (score ≥ 2+) was observed in 14/164 (8.5%) patients. No correlation between MAGE-A10 and NY-ESO-1 expression and tumor size, tumor grade, Ki-67 and lymph nodes status was detectable. MAGE-A10 expression was significantly associated with ER-negative (P = 0.002), PR-negative (P = 0.002) and HER-2-negative (P = 0.044) tumors. We clearly showed that MAGE-A10 is frequently expressed in the group of TN patients, where the majority (85.7%) of tumors express this CTA. Because of limited therapeutic options for the triple-negative breast cancer, the frequent expression of MAGE-A10 CTA in these cancers may offer the opportunity for a much needed additional treatment for this group of patients.

Prognostic role of metastasis tumor antigen 1 in patients with ovarian cancer: a clinical study

In this study, we investigated the prognostic value of metastasis tumor antigen 1 expression in 81 untreated patients with ovarian cancer. The expression of metastasis tumor antigen 1 was evaluated by immunohistochemistry, and staining was analyzed in relation to clinicopathologic variables, disease-free survival, and overall survival. High expression of metastasis tumor antigen 1 was found to be associated with advanced stage (I/II versus III/IV, P = .02) and with worse response to first-line treatment (P = .03). Cases with high metastasis tumor antigen 1 expression showed a lower disease-free survival compared with cases with low expression (P = .02). In multivariate analysis of disease-free survival, metastasis tumor antigen 1 overexpression retained an independent negative prognostic role (P= .04), when considered together with histotype, stage of disease, residual tumor at surgery, and chemosensitivity. The evaluation of the prognostic relevance of metastasis tumor antigen 1 in late-stage disease showed that overexpression was a prognostic factor for poor disease-free survival and overall survival in this subset of patients, in both univariate and multivariate models. These findings indicate that metastasis tumor antigen 1 overexpression can be used as a predictor of clinical outcome in patients with ovarian cancer and therefore may represent a new prognostic marker.

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection

The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay. We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT. In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression. Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Vaccination With OK-432 Followed by TC-1 Tumor Lysate Leads to Significant Antitumor Effects

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

Expression of cancer testis (CT) antigens in human BRCA-associated breast cancers: Potential targets for immunoprevention?

e11098 Background: Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets which are expressed in early cancers, in situ or invasive, but absent in normal breast epithelium of these high-risk individuals could provide the basis for the development of effective immunoprohylactic strategies. Cancer Testes (CT) antigens are potential candidates because their expression is restricted to tumors and accumulating data suggest that they play important roles in cellular proliferation, stem cell function and carcinogenesis of at least some tumors. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers. Methods: Archived breast cancer tissues (n=26) as well as morphologically normal breast tissues (n=7) from women carrying deleterious BRCA 1 and or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10 and GAGE. Results: CT antigens were expressed in 61.5% (16/26) of BRCA-associated cancers. 50% (13/26) of tumors expressed two or more CT antigens; three cancers expressed all seven CT antigens. CT antigens were expressed across all tumor types and stages including DCIS. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (p<0.01). Conclusions: We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.

Abstract 2858: A novel mouse model for cutaneous T-cell lymphoma reveals a role for IL-15 and activity of a new oral HDAC inhibitor

Abstract Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma representing a spectrum of diseases composed of malignant clonal CD4 (+) helper T lymphocytes. The etiology of CTCL remains to be elucidated and no animal models for CTCL are known. We quantified expression of IL-15 in patients with CTCL and demonstrated aberrant overexpression of IL-15 compared to normal CD4+ T cells (Fold increase = 7.476 ± 1.788 N=3, P=0.0073), that showed direct correlation with the stage of the disease (Fold increase in IL-15 in Stage I patients vs. Stage III patients = 3.283 ± 0.8246 vs. 7.476 ± 1.788, N=3 each, P=0.02). We engineered a transgenic (tg) mouse to constitutively and globally overexpress murine IL-15. These mice develop alopecia and cutaneous lesions in all mice within 6 weeks of birth, characterized by erythematous plaques/patches, erythroderma, pruritis and early death compared to wild type (WT) controls (49 weeks vs. 100 weeks, P=0.0003). Skin lesions showed an infiltration of atypical lymphocyte in the dermis and throughout the epidermis including the formation of classic Pautrier's microabscesses as seen in human CTCL. Immunophenotypic analysis of the skin T-cells from these mice revealed ∼25 fold increase in CD3+ T-cells compared to WT littermates. Intradermal CD4+ cells from CTCL mice were predominantly CD62L- and significantly higher than WT mice (P=0.007). Immunohistochemistry showed high expression of cutaneous lymphocyte antigen (CLA) and CCR4 on skin sections of CTCL mice, consistent with a skin-specific homing pattern as seen in human CTCL. Cells obtained from the skin of these IL-15tg CTCL mice could be successfully engrafted into skin of SCID recipient mice with reproducible histological lesions. Consistent with human studies, CTCL mice exhibited significantly increased levels of Hdac-1 in CD4+ T cells when compared to their WT counterparts (P = 0.0013). To investigate the importance of IL-15 in this process, we cultured sorted mouse WT (CD3+CD4+) T-cells in medium supplemented with 100ng/ml IL-15 and observed a significant increase in Hdac-1 expression at the mRNA and protein level within 10 days of culture, compared to freshly isolated WT CD4+ T cells (N=3 each, p =0.02). In a randomized, placebo-controlled trial of an orally administered novel HDAC inhibitor, AR-42 (Arno Therapeutics, Inc, Parsippany, NJ; Sargeant AM et al, Cancer Research, 2008) to IL-15 tg mice with CTCL, we observed an improvement in skin lesions and a marked reduction in T-cell dermal infiltrate as early as 12 days post-treatment. In conclusion, we provide evidence for a reproducible in vivo mouse model of spontaneous CTCL that appears to mimic the human condition. Further, we provide evidence for a role of aberrant IL-15 expression in the genesis of both human and experimental CTCL, and alterations in Hdac-1 expression that may to serve as an appropriate target for a novel oral HDAC inhibitor now in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2011-2858

Expression of cancer testis antigens in human BRCA-associated breast cancers: potential targets for immunoprevention?

Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers. Archived breast cancer tissues (n=26) as well as morphologically normal breast tissues (n=7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE. CT antigens were expressed in 16/26 (61.5%, 95% CI 43-80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P=0.003). We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.

Molecular profile and cellular characterization of human bone marrow mesenchymal stem cells: Donor influence on chondrogenesis

Background The use of autologous or allogenic stem cells has recently been suggested as an alternative therapeutic approach for treatment of cartilage defects. Bone marrow mesenchymal stem cells (BM-MSCs) are well-characterized multipotent cells that can differentiate into different cell types. Understanding the potential of these cells and the molecular mechanisms underlying their differentiation should lead to innovative protocols for clinical applications. The aim of this study was to evaluate the usefulness of surface antigen selection of BM-MSCs and to understand the mechanisms underlying their differentiation. Methods MSCs were isolated from BM stroma and expanded. CD105+ subpopulation was isolated using a magnetic separator. We compared culture-expanded selected cells with non-selected cells. We analyzed the phenotypic profiles, the expression of the stem cell marker genes Nanog, Oct3/4, and Sox2 and the multi-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic). The multi-lineage differentiation was confirmed using histochemistry, immunohistochemistry and/or real-time polymerase chain reaction (qPCR) techniques. Results The selected and non-selected cells displayed similar phenotypes and multi-lineage differentiation potentials. Analyzing each cell source individually, we could divide the six donors into two groups: one with a high percentage of CD29 (β1-integrin) expression (HL); one with a low percentage of CD29 (LL). These two groups had different chondrogenic capacities and different expression levels of the stem cell marker genes. Conclusions This study showed that phenotypic profiles of donors were related to the chondrogenic potential of human BM-MSCs. The chondrogenic potential of donors was related to CD29 expression levels. The high expression of CD29 antigen seemed necessary for chondrogenic differentiation. Further investigation into the mechanisms responsible for these differences in BM-MSCs chondrogenesis is therefore warranted. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous human BM-MSCs for articular cartilage repair.

High Expression of the Very Late Antigen (VLA)-4 (CD49d) Integrin Predicts for Reduced Risk of Relapse and Better Outcome in Pediatric Acute Myeloid Leukemia (AML): A Report From the Children's Oncology Group.

Abstract 1592▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-618 BackgroundPrevious studies highlighted the importance of the cell adhesion molecule, VLA-4, for chemoresistance and minimal residual disease (MRD) in AML, suggesting promise as therapeutic target. By comparison, the prognostic role of VLA-4 in AML remains controversial with retrospective studies implying either adverse or favorable prognosis. Therefore, we prospectively evaluated VLA-4 expression in participants of a recent Children's Oncology Group (COG) AML pilot protocol. MethodsCOG-AAML03P1 enrolled 340 newly diagnosed children (aged 1 month - 21 years) with de novo non-acute promyelocytic AML, excluding those with Down syndrome, and tested the feasibility of combining gemtuzumab ozogamicin (GO) with intensive induction chemotherapy followed by GO-containing intensification therapy or matched related donor stem cell transplantation; 216 patients submitted diagnostic marrow specimens for flow cytometric determination of VLA-4 expression that was then correlated with patient demographics, laboratory characteristics, and clinical outcome. Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa; n=73), high risk (-5/5q-, monosomy 7, or FLT3/ITD with high allelic ratio; n=25), or standard risk (all other patients with cytogenetic/molecular data; n=101); 17 patients had insufficient data for risk classification. ResultsAmong the 216 diagnostic specimens, the mean fluorescence intensity (MFI) of VLA-4 expression varied over 35-fold from a baseline of 30 to 1110 (median, 219.5). Patients with high VLA-expression (>median MFI) were younger (p<0.001), had a lower prevalence of FLT3/ITD (p=0.002). Presence of extramedullary disease (EMD, chloroma or CNS involvement) was significantly higher in patients with high VLA-4 expression (16% vs. 5%, p=0.013), where 17/22 (77%) of patients with EMD had high VLA-4 expression. We initially inquired whether VLA-4 expression as a continuous variable correlated with disease outcome. We demonstrated that over the range of VLA-4 expression levels, every 10-unit increase in VLA-4 MFI corresponded to a 2% decrease in relapse risk (RR; p=0.023) and 2% increase in disease-free survival (DFS) from end of induction I (p=0.015). We subsequently divided the study patients into two cohorts based on median MFI and determined the clinical outcome per median VLA-4 MFI for the entire cohort as well as in specific risk groups (i.e., high risk, low risk, and standard risk). In the evaluation of the entire cohort, patients with high VLA-4 expression had a significantly superior DFS (67% vs 48%, p=0.023) and lower RR (24% vs 44%, p=0.011) compared to those with lower VLA-4 expression. In subgroup analyses, high VLA-4 expression was associated with significantly superior DFS (69% vs 34%, p=0.011) and lower RR (26% vs 61%, p=0.009) in patients with standard-risk AML but not in patients with high or low risk disease. In multivariate Cox regression analyses that included age, cytogenetics, and molecular prognostic factors, low VLA-4 expression remained significantly associated with elevated RR (hazard ratio for low VLA-4: 2.25, p=0.011). Finally, we determined the impact of MRD in the context of VLA-4 expression. The prevalence of MRD was similar for patients with low or high VLA-4 expression (29% vs. 25%, p=0.70). In patients with low VLA-4 expression, those with MRD had a RR of 75% compared to that of 35% for the MRD negative cohort (p=0.005). Corresponding DFS was 19% and 54% for those with and without MRD (p=0.018), a difference mainly attributable to increased RR. In patients with high VLA-4 expression, those with MRD had a RR of 31% vs. 23% for the MRD-negative patients (p=0.28) with a corresponding DFS of 46% and 75% for the MRD positive and negative patients (p=0.014), a difference mainly explained by higher treatment-related mortality in MRD positive patients. ConclusionThis study demonstrates significant heterogeneity of VLA-4 expression in pediatric AML and its association with EMD and clinical outcome. This study further demonstrates that VLA-4 expression is an independent prognostic factor for clinical outcome and can identify the risk status in patients with no identifiable cytogenetic or molecular risk factors. DisclosuresNo relevant conflicts of interest to declare.

Relationship Between CD56 Antigen Expression and Quantification of MDR1 Gene Expression in Patients with De Novo Acute Myeloid Leukemia(AML).

Abstract 4677 IntrodutionThe expression of CD56 antigen and over-expression of the multidrug resistance gene 1 (MDR1) seems to confer the poor therapeutic outcome to AML. The aim of this study is to investigate the relationship between CD56 antigen expression and MDR1 gene expression in patients with de novo AML and explore the indicating effect of these two factors on clinical drug resistance. Patients and methodsA real-time quantitative reverse transcriptase polymerase chain reaction method was established for detecting MDR1 expression levels and a three-color flow cytometry analysis with CD45/SSC gating was used to examine CD56 antigen expression in 79 patients with de novo AML. ResultsCD56 antigen was recorded in 19 out of 79 cases (24.1%) and particularly in those with M5 subtype and t(8;21) AML. Moreover, CD56 expression was significantly associated with unfavorable cytogenetic abnormalities (P< 0.05). Significantly higher percentage (57.1%, 4/7) of patients with t(8;21) demonstrated CD56 expression than those with favorable cytogenetic abnormalities (P< 0.05). CD56+ AML patients had higher incidence of splenohepatomegalia and level of lactate dehydrogenase than CD56- patients (P< 0.05). The median expression level of MDR1 was statistically higher in CD56+ AML patients than that in CD56- cases (P< 0.001) and 89.5% (17/19) CD56+ AML patients were found with high MDR1 expression. The CR rate in high MDR1 / CD56+ AML patients was significantly lower than that in low MDR1/ CD56- cases. (58.8% vs 89.2%, P< 0.01). ConclusionsThere is a linear correlation between MDR1 and CD56 expression in AML. This relationship may explain why CD56 expression is related to a poor prognosis in AML. Therefore both with high MDR1 expression level and CD56 antigen expression can identify AML patients with unfavorable outcome. Disclosures:No relevant conflicts of interest to declare.

Therapy of B-Cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, Is Mediated through Hyperactivation of ERK and JNK MAP Kinase Signaling Pathways.

Abstract 3738Poster Board III-674 IntroductionHLA-DR is currently under investigation as a target for antibody (mAb) therapy of B-cell malignancies. IMMU-114, a humanized IgG4 form of the murine anti-HLA-DR mAb, L243, recognizes a conformational epitope in the a-chain of HLA-DR. We have previously reported that IMMU-114 lacks effector-cell functions while displaying potent antitumor activity against B-cell lymphomas in vitro and in vivo. Here, we further investigated the cytotoxic effects of IMMU-114 on leukemia, lymphoma, and multiple myeloma (MM) cell lines, as well as clinical specimens of CLL, and explored the signaling pathways involved. MethodsBinding and cytotoxicity of IMMU-114 was examined on a panel of NHL, AML, MCL, ALL, and CLL cell lines. Induction of apoptosis (Annexin V binding), ROS generation (dihydoehidium staining), changes in mitochondrial membrane potential (ψM, TMRE staining) and effects on the ERK1/2 and JNK pathways (immunoblot analyses) were evaluated on selected cell lines. The effects of various inhibitors on IMMU-114-mediated apoptosis also were determined. Studies were performed in tumor-bearing SCID mice. ResultsHigh expression of HLA-DR was detected on the cell surface of all MCL, ALL, HC, CLL, and NHL cell lines tested, as well as 2/3 AML and 5/6 MM cell lines. IMMU-114 was cytotoxic to 2/2 MCL, 2/2 CLL, 2/4 ALL, 1/1 HC, 2/2 NHL and 2/2 MM cell lines without any added anti-Fc second crosslinking antibody. IMMU-114 was consistently more cytotoxic than anti-CD20 mAbs on these cell lines, as well as on rituximab-resistant variants of Raji and SU-DHL-4. Despite positive staining, AML cell lines, Kasumi-3 and GDM-1, were not killed by IMMU-114. Four of 6 CLL patient samples expressed HLA-DR at markedly higher levels than CD20, with the remaining 2 showing similar HLA-DR and CD20 expression. Approximately 60% cytotoxicity was obtained after incubation with IMMU-114 in CLL patient cells. Induction of apoptosis as well as changes in ψM and ROS correlated with sensitivity of cell lines to IMMU-114-mediated cytotoxicity. Time-course analyses demonstrated that IMMU-114 induced approximately 46% change of ψM and 24% enhancement of ROS in as little as 30 min in Raji cells. These changes were abrogated in the presence of ROS inhibitor, N-acetyl cysteine. Immunoblot analyses revealed that IMMU-114 induces phosphorylation of ERK and JNK mitogen-activated protein (MAP) kinases in all cells defined as IMMU-114-sensitive by the cytotoxicity assays, but not IMMU-114-resistant cell lines. The p38 pathway was found to be constitutively active in these cell lines. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis, although the inhibition was not complete when any single inhibitor was used. This could be due to activation of multiple pathways, since inhibition of 2 or more pathways by specific inhibitors abolishes the apoptosis induced by IMMU-114. Activation (5-fold increases of phosphorylation) of ERK1/2 and JNK signaling pathways also was observed in CLL patient samples following IMMU-114 incubation. Further studies revealed caspase-independent apoptosis and activation of apoptosis-inducing factor (AIF) mediated by IMMU-114. The therapeutic activity of IMMU-114 was evaluated in vivo in 3 NHL models, WSU-FSCCL, Namalwa, and Raji. IMMU-114 was more effective than anti-B-cell mAbs such as rituximab, in all models. For example, in WSU-FSCCL, IMMU-114 treatment yielded 100% long-term survival. Although rituximab also induced significant responses compared to untreated mice (median survival time of 87.5 vs. 36 days for untreated controls, P<0.0001), no long-term survivors were seen. ConclusionIMMU-114 is cytotoxic to a variety of hematological malignancies, and its potent activity can be related to high antigen expression of target cells and hyperactivation of ERK and JNK pathways upon binding to HLA-DR. Clinical evaluation of IMMU-114 is planned. Disclosures:Gupta:Immunomedics Inc.: Employment. Stein:Garden State Cancer Center: Employment. Cardillo:Immunomedics Inc.: Employment. Chang:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Human leukocyte antigen class I expression is an independent prognostic factor in advanced ovarian cancer resistant to first-line platinum chemotherapy

Background:Loss of HLA class I is important in ovarian cancer prognosis but its role as a prognostic indicator in relation to therapy remains unproven. We studied the prognostic potential of this antigen and its significance in relation to platinum therapy.Methods:A total of 157 primary ovarian cancers were assessed for HLA class I immunohistochemically and linked to a comprehensive database of clinicopathological variables, treatment details, and platinum sensitivity.Results:Tumours expressing high levels of HLA class I had significantly improved survival (P=0.044). There was a 19-month difference in the median overall survival between tumours with high and low antigen expression. HLA class I antigen expression, stage, and platinum sensitivity were independently predictive of prognosis on multivariate analysis. HLA class I antigen was shown to be expressed at higher levels in patients with good overall survival in platinum-resistant patients (P=0.042). HLA class I significantly correlated with overall survival on multivariate analyses (P=0.034).Conclusion:Low-level HLA class I expression is an independent prognostic indicator of poor clinical outcome in ovarian cancer. The survival advantage of patients with platinum-resistant tumours expressing high levels of HLA class I suggests that immunotherapy may be of use in these ovarian cancers resistant to standard chemotherapy.

Immunohistochemical Expression of Prostate Stem Cell Antigen in Cystoprostatectomies with Incidental Prostate Cancer

High expression of prostate stem cell antigen (PSCA) has been shown to be associated with adverse prognostic features in clinically-diagnosed prostate cancer. The aim of this study is to analyze PSCA expression in cystoprostatectomies with incidental prostate carcinoma (PCa). PSCA expression was evaluated immunohistochemically in normal-looking epithelium (NEp), high-grade prostatic intraepithelial neoplasia (HGPIN) and pT2a Gleason score 6 acinar adenocarcinoma. The evaluation was carried out on 20 cystoprostatectomies (CyPs) with incidental PCa from men with bladder urothelial carcinoma (UC), and 20 radical prostatectomies (RPs) with hormonally untreated PCa from men with clinically detected PCa. Ki-67 was also investigated. The percentages of PSCA positive cells in HGPIN were significantly higher than in NEp (NEp: CyP, mean 2.92%+/-standard deviation 6.26%; RP, 3.5%+/-6.46%. HGPIN: CyP, 13.67%+/-12.78%; RP, 14.67%+/-11.34%) (p<0.001). The proportions of positive cells in PCa were greater than in HGPIN (CyP, 20.25%+/-15.96%; RP, 22.58%+/-13.67%) (p<0.001). For Ki-67 labeling, the proportions of positive nuclei in the CyPs significantly increased from NEp through HGPIN to PCa. A similar trend was seen in the RPs. In the CyPs the percentages of PSCA and Ki67 positive cells were lower than in the RPs, the differences between the CyP and RP compartments being not statistically significant. Our findings suggest that PSCA is a marker associated with neoplastic transformation of prostate cells, both in CyPs and RPs. However, there are no significant differences between CyPs with incidental prostate carcinoma and RPs with clinically diagnosed cancer.

High Aldehyde Dehydrogenase Activity: A Novel Functional Marker of Murine Prostate Stem/Progenitor Cells

Abstract We have shown previously that prostatic stem/progenitor cells can be purified from isolated prostate ducts, based on their high expression of the Sca-1 surface antigen. We now report that high levels of aldehyde dehydrogenase (ALDH) activity are present in a subset of prostate epithelial cells that coexpress a number of antigens found on stem/progenitor cells of other origins (CD9, Bcl-2, CD200, CD24, prominin, Oct 3/4, ABCG2, and nestin). Almost all of these cells expressing high levels of ALDH activity also express Sca-1 and a third of them express high levels of this antigen. The cells with high levels of ALDH activity have greater in vitro proliferative potential than cells with low ALDH activity. Importantly, in an in vivo prostate reconstitution assay, the cells expressing high levels of ALDH activity were much more effective in generating prostatic tissue than a population of cells with low enzymatic activity. Thus, a high level of ALDH activity can be considered a functional marker of prostate stem/progenitor cells and allows for simple, efficient isolation of cells with primitive features. The elucidation of the role of ALDH in prostate stem/progenitor cells may lead to the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia. Disclosure of potential conflicts of interest is found at the end of this article.