Knutson, Keith L.

Abstract To reveal biologic mechanisms underlying clinical outcome of high-grade serous (HGS) epithelial ovarian carcinomas (EOC), we evaluated the association between tumor epigenetic changes and time to recurrence (TTR). We assessed methylation at approximately 450,000 genome-wide CpGs in tumors of 337 Mayo Clinic (Rochester, MN) patients. Semi-supervised clustering of discovery (n = 168) and validation (n = 169) sets was used to determine clinically relevant methylation classes. Clustering identified two methylation classes based on 60 informative CpGs, which differed in TTR in the validation set [R vs. L class, P = 2.9 × 10−3, HR = 0.52; 95% confidence interval (CI), 0.34–0.80]. Follow-up analyses considered genome-wide tumor mRNA expression (n = 104) and CD8 T-cell infiltration (n = 89) in patient subsets. Hypomethylation of CpGs located in 6p21.3 in the R class associated with cis upregulation of genes enriched in immune response processes (TAP1, PSMB8, PSMB9, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DOA), increased CD8 T-cell tumor infiltration (P = 7.6 × 10−5), and trans-regulation of genes in immune-related pathways (P = 1.6 × 10−32). This is the most comprehensive assessment of clinical outcomes with regard to epithelial ovarian carcinoma tumor methylation to date. Collectively, these results suggest that an epigenetically mediated immune response is a predictor of recurrence and, possibly, treatment response for HGS EOC. Cancer Res; 74(11); 3084–91. ©2014 AACR.

Abstract Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer–bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1–negative tumors). Cancer Res; 74(11); 2974–85. ©2014 AACR.

Abstract The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22–1.64; P = 5.7 × 10−6], rs791587 (HR, 1.36; 95% CI, 1.17–1.57; P = 6.2 × 10−5), rs2476491 (HR, = 1.40; 95% CI, 1.19–1.64; P = 5.6 × 10−5), and rs10795763 (HR, 1.35; 95% CI, 1.17–1.57; P = 7.9 × 10−5), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54–0.82; P = 9.3 × 10−5) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer. Cancer Immunol Res; 2(4); 332–40. ©2014 AACR.

Abstract Background: Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related death in females and leading gynecologic cause of cancer-related death. Despite the identification of a number of serum biomarkers, methods to identify early-stage disease and predict prognosis remain scarce. We have evaluated two biologically connected serum biomarkers, serum leukocyte protease inhibitor (SLPI) and progranulin (PGRN). Methods: Two-hundred frozen plasma samples were acquired from the Mayo Clinic Biospecimen Repository for Ovarian Cancer Research. Samples were obtained from 50 patients with benign conditions, 50 with American Joint Committee on Cancer (AJCC) stage I and II EOC, and 100 with AJCC stage III and IV EOC. Samples were obtained before surgical resection of a mass and were analyzed for absolute levels of SLPI and PGRN using ELISA assays. Receiver-operator characteristic curves were generated for SLPI and PGRN. Median follow-up was 48 months. Results: Absolute levels of SLPI were significantly elevated in patients with EOC compared with benign disease and predicted the presence of EOC (AUC of 0.812; P = 0.04); SLPI remained elevated in the subset of patients with normal CA-125. PGRN levels were not significantly increased in patients with early-stage or late-stage EOC as a whole, but an increase in PGRN levels was associated with decreased overall survival in advanced EOC. Conclusions: SLPI levels are elevated in EOC, and SLPI shows promise as a diagnostic biomarker for patients with both elevated and normal CA-125 levels. An increase in PGRN is associated with decreased overall survival. Impact: SLPI is elevated in EOC and warrants investigation in a screening study in women at risk for EOC. Cancer Epidemiol Biomarkers Prev; 22(10); 1730–5. ©2013 AACR.

Abstract Patients with HER-2/neu–expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2–binding peptide p369–377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu–overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369–377 was processed from larger HER-2/neu–derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369–377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373–382 (SLAFLPESFD), bound HLA-A2 stronger than did p369–377. CTLs specific for p373–382 recognized both p373–382 and p369–377 complexed with HLA-A2. CTLs specific for p373–382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369–377. Conversely, CTLs specific for p369–377 recognized p373–382. Peptide p373–382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

Abstract Background: Ovarian cancer (OC) is the leading cause of gynecologic cancer death in the United States. Survival in advanced OC is highly variable; some patients are cured of their disease with a combination of surgery and systemic chemotherapy, while the majority of patients develop recurrence and ultimately die of metastatic OC. While markers of poor prognosis within advanced OC exist, they have not translated to changes in therapy or outcomes. Vascular growth factors are important soluble mediators of tumor growth, vasculogenesis and angiogenesis that may be targeted therapeutically. We therefore explored the association between plasma levels of vascular growth factors and clinical outcomes in advanced OC. Methods: We enrolled 200 patients with OC or benign surgical conditions from the Mayo Ovarian SPORE Biospecimens Core. Presurgical plasma samples from patients with advanced OC (AJCC Stage III and IV, n = 100), early stage OC (Stage I and II, n = 50), and benign surgical conditions (n = 50) were assayed for vascular growth factors via multiplex electrochemiluminescence and by ELISA. Differences in plasma levels between benign, early, and advanced OC patient groups were assessed using plate-adjusted linear models. Associations between plasma levels of vascular growth factors and overall survival in advanced OC patients were assessed by Cox proportional hazards models. Results: Both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) levels were increased in patients with advanced OC (mean ng/mL = 130.8, p = 0.004; mean ng/mL = 20.1, p = 0.025, respectively) compared to patients with early stage (mean ng/mL = 75.1 and 19.0 respectively) and benign conditions (mean ng/mL = 86.8 and 15.0, respectively). An increase in PlGF levels was associated with poor overall survival in patients with advanced OC (HR = 2.52, p = 0.005), while increase in VEGF levels were of borderline significance (HR = 1.21, p = 0.061) Conclusions: Soluble PlGF and VEGF are increased in the setting of advanced OC. Increased plasma PlGF is strongly linked to poor prognosis, while VEGF levels have a borderline association with prognosis. These data provide a rationale for therapeutic targeting of both VEGF and PlGF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1382. doi:1538-7445.AM2012-1382

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. 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 3341. doi:10.1158/1538-7445.AM2011-3341

Abstract CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.

AbstractBreast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial–mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer. Cancer Res; 71(13); 4707–19. ©2011 AACR.