Description: Purpose: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers. Experimental Design: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial. Results: We identified a distinct "reactive stroma" gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro . Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy. Conclusions: Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer. Clin Cancer Res; 21(13); 2941–51. ©2015 AACR .

Description: Purpose: We conducted a randomized phase III study to determine whether patients with early breast cancer would benefit from the addition of capecitabine (X) to a standard regimen of doxorubicin (A) plus cyclophosphamide (C) followed by docetaxel (T). Experimental Design: Treatment comprised eight cycles of AC-〉T (T dose: 100 mg/m 2 on day 1) or AC-〉XT (X dose: 825 mg/m 2 twice daily, days 1–14; T dose: 75 mg/m 2 on day 1). The primary endpoint was 5-year disease-free survival (DFS). Results: Of 2,611 women, 1,304 were randomly assigned to receive AC-〉T and 1,307 to receive AC-〉XT. After a median follow-up of 5 years, the study failed to meet its primary endpoint [HR, 0.84; 95% confidence interval (CI), 0.67–1.05; P = 0.125]. A significant improvement in overall survival, a secondary endpoint, was seen with AC-〉XT versus AC-〉T (HR, 0.68; 95% CI, 0.51–0.92; P = 0.011). There were no unexpected adverse events. Of patients with estrogen receptor (ER)–positive/HER2-negative disease, 70% of whom were node-positive, 26% and 59% had tumors with a centrally assessed Ki-67 score of 〈10% or 〈20%, respectively, and only 17 (2%) and 53 (6%) DFS events, respectively, occurred in these groups at 7 years. Conclusions: The very low event rate in patients with ER-positive, low Ki-67 cancers, regardless of nodal status, strongly suggests that these patients should not be enrolled in adjuvant trials that assess 5-year DFS rates and that central Ki-67 analyses can identify these patients. Clin Cancer Res; 21(19); 4305–11. ©2015 AACR .

Description: Purpose: We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. Experimental Design: We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications. Results: Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the NAPRT1 promoter identified tumor-specific sites of NAPRT1 DNA methylation and enabled the development of a quantitative methylation-specific PCR (QMSP) assay suitable for use on archival formalin-fixed paraffin-embedded tumor tissue. Conclusions: Tumor-specific promoter hypermethylation of NAPRT1 inactivates one of two NAD salvage pathways, resulting in synthetic lethality with the coadministration of a NAMPT inhibitor. NAPRT1 expression is lost due to promoter hypermethylation in most cancer types evaluated at frequencies ranging from 5% to 65%. NAPRT1-specific immunohistochemical or DNA methylation assays can be used on archival formalin paraffin-embedded cancer tissue to identify patients likely to benefit from coadministration of a Nampt inhibitor and nicotinic acid. Clin Cancer Res; 19(24); 6912–23. ©2013 AACR .

Description: Purpose: Glioblastoma is a devastating, incurable disease with few known prognostic factors. Here, we present the first genome-wide survival and validation study for glioblastoma. Experimental Design: Cox regressions for survival with 314,635 inherited autosomal single-nucleotide polymorphisms (SNP) among 315 San Francisco Adult Glioma Study patients for discovery and three independent validation data sets [87 Mayo Clinic, 232 glioma patients recruited from several medical centers in Southeastern United States (GliomaSE), and 115 The Cancer Genome Atlas patients] were used to identify SNPs associated with overall survival for Caucasian glioblastoma patients treated with the current standard of care, resection, radiation, and temozolomide (total n = 749). Tumor expression of the gene that contained the identified prognostic SNP was examined in three separate data sets (total n = 619). Genotype imputation was used to estimate hazard ratios (HR) for SNPs that had not been directly genotyped. Results: From the discovery and validation analyses, we identified a variant in single-stranded DNA-binding protein 2 ( SSBP2 ) on 5q14.1 associated with overall survival in combined analyses (HR, 1.64; P = 1.3 x 10 –6 ). Expression of SSBP2 in tumors from three independent data sets also was significantly related to patient survival ( P = 5.3 x 10 –4 ). Using genotype imputation, the SSBP2 SNP rs17296479 had the strongest statistically significant genome-wide association with poorer overall patient survival (HR, 1.79; 95% CI, 1.45-2.22; P = 1.0 x 10 –7 ). Conclusion: The minor allele of SSBP2 SNP rs17296479 and the increased tumor expression of SSBP2 were statistically significantly associated with poorer overall survival among glioblastoma patients. With further confirmation, previously unrecognized inherited variations influencing survival may warrant inclusion in clinical trials to improve randomization. Unaccounted for genetic influence on survival could produce unwanted bias in such studies. Clin Cancer Res; 18(11); 3154–62. ©2012 AACR .

Description: CXC chemokines and their cognate receptors have been implicated widely in cancer pathogenesis. In this study, we report a critical causal relationship between CXCR6 expression and tumorigenesis in the setting of human hepatocellular carcinoma (HCC). Among the CXC chemokine receptors, only CXCR6 was detected in all the hepatoma cell lines studied. Moreover, in HCC tissue, CXCR6 expression was significantly higher than in noncancerous liver tissues. Reduction of CXCR6 or its ligand CXCL16 in cancer cells reduced cell invasion in vitro and tumor growth, angiogenesis, and metastases in vivo. Importantly, loss of CXCR6 led to reduced Gr-1+ neutrophil infiltration and decreased neoangiogenesis in hepatoma xenografts via inhibition of proinflammatory cytokine production. Clinically, high expression of CXCR6 was an independent predictor of increased recurrence and poor survival in HCCs. Human HCC samples expressing high levels of CXCR6 also contained an increased number of CD66b+ neutrophils and microvessels, and the combination of CXCR6 and neutrophils was a superior predictor of recurrence and survival than either marker used alone. Together, our findings suggest that elevated expression of CXCR6 promotes HCC invasiveness and a protumor inflammatory environment and is associated with poor patient outcome. These results support the concept that inhibition of the CXCR6–CXCL16 pathway may improve prognosis after HCC treatment. Cancer Res; 72(14); 3546–56. ©2012 AACR.

Description: Purpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. Experimental Design: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo . In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations. Clin Cancer Res; 19(24); 6976–86. ©2013 AACR .

Description: HHLA2 is a newly identified B7 family member that modulates T-cell functions through interaction with TMIGD2 and possibly a second receptor, with coinhibition in two studies and costimulation in one study. HHLA2 is expressed on a variety of human cancers, and its coinhibitory function makes it a candidate for cancer immunotherapy. Clin Cancer Res; 21(10); 2201–3. ©2015 AACR . See related article by Janakiram et al., p. 2359

Description: 14-3-3ζ is overexpressed in more than 40% of breast cancers, but its pathophysiologic relevance to tumorigenesis has not been established. Here, we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter-driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors, whereas control mice did not. Whey acidic protein promoter-driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared with MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling, leading to increased miR-221 transcription, which inhibited p27 CDKI translation and, consequently, promoted cell proliferation. Importantly, this 14-3-3ζ–miR-221–p27 proliferation axis is also functioning in breast tumors in patients and is associated with high-grade cancers. Taken together, our findings show that overexpression of 14-3-3ζ has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation. Cancer Res; 74(1); 363–73. ©2013 AACR.

Description: While showing promise, vaccination strategies to treat cancer require further optimization. Likely barriers to efficacy involve cancer-associated immunosuppression and peripheral tolerance, which limit the generation of effective vaccine-specific cytotoxic T lymphocytes (CTL). Because CD4+ T cells improve CTL responsiveness, next-generation vaccines include helper epitopes. Here, we demonstrate in mice how CD4+ T-cell help optimizes the CTL response to a clinically relevant DNA vaccine engineered to combat human papillomavirus–expressing tumors. Inclusion of tumor-unrelated helper epitopes greatly increased CTL priming, effector, and memory T-cell programming. CD4+ T-cell help optimized the CTL response in all these aspects via CD27/CD70 costimulation. Notably, administration of an agonistic CD27 antibody could largely replace helper epitopes in promoting primary and memory CTL responses, acting directly on CD8+ T cells. CD27 agonism improved efficacy of the vaccine without helper epitopes, more so than combined PD-1 and CTLA-4 blockade. Combining CD27 agonism with CTLA-4 blockade improved vaccine-induced CTL priming and tumor infiltration, but only combination with PD-1 blockade was effective at eradicating tumors, thereby fully recapitulating the effect of CD4+ T-cell help on vaccine efficacy. PD-1 blockade alone did not affect CTL priming or tumor infiltration, so these results implied that it cooperated with CD4+ T-cell help by alleviating immune suppression against CTL in the tumor. Helper epitope inclusion or CD27 agonism did not stimulate regulatory T cells, and vaccine efficacy was also improved by CD27 agonism in the presence of CD4+ T-cell help. Our findings provide a preclinical rationale to apply CD27 agonist antibodies, either alone or combined with PD-1 blockade, to improve the therapeutic efficacy of cancer vaccines and immunotherapy generally. Cancer Res; 76(10); 2921–31. ©2016 AACR.

Description: Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN-γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies. Cancer Res; 72(17); 4417–28. ©2012 AACR.