CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients.

Regulatory T cells (T(regs)) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25(+) FoxP3(+) T(regs) in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration-approved CD25-blocking monoclonal antibody daclizumab with regard to human T(reg) survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25(high) CD45RA(neg) T(regs). Moreover, daclizumab-treated CD45RA(neg) T(regs) lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on T(regs) in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in T(regs) in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms T(regs) in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated T(regs) while avoiding autoimmunity.

Frequent genetic abnormalities of the PI3K/AKT pathway in primary ovarian cancer predict patient outcome.

Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.

Functional unresponsiveness and replicative senescence of myeloid leukemia antigen-specific CD8+ T cells after allogeneic stem cell transplantation.

PURPOSE: The therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with myeloid malignancies has been attributed in part to a graft-versus-leukemia effect that is dependent on donor T lymphocytes. CD8(+) T-cell responses to MHC class I-restricted tumor epitopes, not just allogeneic antigens, may help mediate antileukemia effects after HSCT, but the specificity and function of such cells are not completely understood. EXPERIMENTAL DESIGN: We examined the diversity, phenotype, and functional potential of leukemia-associated antigen-specific CD8(+) T cells in patients with myeloid leukemia following allogeneic HSCT. Screening for antigen-specific T cells was accomplished with a peptide/MHC tetramer library. RESULTS: Patients with acute myelogenous leukemia or chronic myelogenous leukemia in remission following HSCT exhibited significant numbers of peripheral blood CD8(+) T cells that recognized varying combinations of epitopes derived from leukemia-associated antigens. However, these cells failed to proliferate, release cytokines, or degranulate in response to antigen-specific stimuli. As early as 2 months after HSCT, CD8(+) T cells from patients were predominantly CD28(-) CD57(+) and had relatively short telomeres, consistent with cellular senescence. CONCLUSIONS: Circulating leukemia-specific CD8(+) T cells are prominent in myeloid leukemia patients after HSCT, but such cells are largely functionally unresponsive, most likely due to replicative senescence. These findings carry important implications for the understanding of the graft-versus-leukemia effect and for the rational design of immunotherapeutic strategies for patients with myeloid leukemias.

Collapse of the CD27+ B-cell compartment associated with systemic plasmacytosis in patients with advanced melanoma and other cancers.

PURPOSE: Disturbed peripheral blood B-cell homeostasis complicates certain infections and autoimmune diseases, such as HIV and systemic lupus erythematosus, but has not been reported in cancer. This study aimed to investigate whether B-cell physiology was altered in the presence of melanoma and other cancers. EXPERIMENTAL DESIGN: Flow cytometry was used to identify phenotypic differences in B cells from patients with melanoma and normal donors. In vitro stimulated B cells were assessed for responsiveness and also used as stimulators of allogeneic T cells in mixed lymphocyte reactions. RESULTS: We show B-cell dysregulation in patients with advanced melanoma (n = 26) and other solid tumors (n = 13), marked by a relative and absolute loss of CD27+ (memory) B cells and associated with an aberrant systemic plasmacytosis. Functionally, B cells from patients with melanoma inefficiently up-regulated immunoregulatory molecules and weakly secreted cytokines in response to CD40 and toll-like receptor 9 agonists. Stimulated B cells from patients induced proliferation of alloreactive CD4+ T cells, but these T cells poorly secreted IFNgamma and interleukin-2. These effects were recapitulated by using purified normal donor CD27(neg) B cells in these same assays, linking the predominance of CD27(neg) B cells in patients with the observed functional hyporesponsiveness. Indeed, B-cell dysfunction in patients strongly correlated with the extent of loss of CD27+ B cells in peripheral blood. CONCLUSIONS: Disturbed B-cell homeostasis is a previously unrecognized feature of patients with advanced melanoma and other cancers and may represent an unanticipated mechanism of immune incompetence in cancer.

High resolution genomic analysis of sporadic breast cancer using array-based comparative genomic hybridization.

INTRODUCTION: Genomic aberrations in the form of subchromosomal DNA copy number changes are a hallmark of epithelial cancers, including breast cancer. The goal of the present study was to analyze such aberrations in breast cancer at high resolution. METHODS: We employed high-resolution array comparative genomic hybridization with 4,134 bacterial artificial chromosomes that cover the genome at 0.9 megabase resolution to analyze 47 primary breast tumors and 18 breast cancer cell lines. RESULTS: Common amplicons included 8q24.3 (amplified in 79% of tumors, with 5/47 exhibiting high level amplification), 1q32.1 and 16p13.3 (amplified in 66% and 57% of tumors, respectively). Moreover, we found several positive correlations between specific amplicons from different chromosomes, suggesting the existence of cooperating genetic loci. Queried by gene, the most frequently amplified kinase was PTK2 (79% of tumors), whereas the most frequently lost kinase was PTK2B (hemizygous loss in 34% of tumors). Amplification of ERBB2 as measured by comparative genomic hybridization (CGH) correlated closely with ERBB2 DNA and RNA levels measured by quantitative PCR as well as with ERBB2 protein levels. The overall frequency of recurrent losses was lower, with no region lost in more than 50% of tumors; the most frequently lost tumor suppressor gene was RB1 (hemizygous loss in 26% of tumors). Finally, we find that specific copy number changes in cell lines closely mimicked those in primary tumors, with an overall Pearson correlation coefficient of 0.843 for gains and 0.734 for losses. CONCLUSION: High resolution CGH analysis of breast cancer reveals several regions where DNA copy number is commonly gained or lost, that non-random correlations between specific amplicons exist, and that specific genetic alterations are maintained in breast cancer cell lines despite repeat passage in tissue culture. These observations suggest that genes within these regions are critical to the malignant phenotype and may thus serve as future therapeutic targets.