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Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014-25. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: Here we assess whether molecular subtyping identifies biological features of tumors that correlate with survival and surgical outcomes of high-grade serous ovarian cancer (HGSOC). Experimental Design: Consensus clustering of pooled mRNA expression data from over 2,000 HGSOC cases was used to define molecular subtypes of HGSOCs. This de novo classification scheme was then applied to 381 Mayo Clinic HGSOC patients with detailed survival and surgical outcome information. Results: Five molecular subtypes of HGSOC were identified. In the pooled dataset, three subtypes were largely concordant with prior studies describing proliferative, mesenchymal, and immunoreactive tumors (concordance > 70%), and the group of tumors previously described as differentiated type was segregated into two new types, one of which (anti-mesenchymal) had downregulation of genes that were typically upregulated in the mesenchymal subtype. Molecular subtypes were significantly associated with overall survival ( P < 0.001) and with rate of optimal surgical debulking (≤1 cm, P = 1.9E-4) in the pooled dataset. Among stage III-C or IV Mayo Clinic patients, molecular subtypes were also significantly associated with overall survival ( P = 0.001), as well as rate of complete surgical debulking (no residual disease; 16% in mesenchymal tumors compared with >28% in other subtypes; P = 0.02). Conclusions: HGSOC tumors may be categorized into five molecular subtypes that associate with overall survival and the extent of residual disease following debulking surgery. Because mesenchymal tumors may have features that were associated with less favorable surgical outcome, molecular subtyping may have future utility in guiding neoadjuvant treatment decisions for women with HGSOC. Clin Cancer Res; 23(15); 4077-85. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value., Experimental Design: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types., Results: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types., Conclusions: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746-55. ©2016 AACR., Competing Interests: R.S.H. is a co-founder of ApoGen Biotechnologies Inc. The other authors have no conflicts of interest to disclose., (©2016 American Association for Cancer Research.)

Purpose: Ovarian cancer has a high recurrence and mortality rate. A barrier to improved outcomes includes a lack of accurate models for preclinical testing of novel therapeutics., Experimental Design: Clinically relevant, patient-derived tumorgraft models were generated from sequential patients and the first 168 engrafted models are described. Fresh ovarian, primary peritoneal, and fallopian tube carcinomas were collected at the time of debulking surgery and injected intraperitoneally into severe combined immunodeficient mice., Results: Tumorgrafts demonstrated a 74% engraftment rate with microscopic fidelity of primary tumor characteristics. Low-passage tumorgrafts also showed comparable genomic aberrations with the corresponding primary tumor and exhibit gene set enrichment of multiple ovarian cancer molecular subtypes, similar to patient tumors. Importantly, each of these tumorgraft models is annotated with clinical data and for those that have been tested, response to platinum chemotherapy correlates with the source patient., Conclusions: Presented herein is the largest known living tumor bank of patient-derived, ovarian tumorgraft models that can be applied to the development of personalized cancer treatment., (©2014 AACR)

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer., Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients., Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00-2.24, P = 0.052)., Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer., (©2011 AACR.)

Purpose: Due to variation of outcome among cases, we sought to examine whether overall survival in ovarian cancer was associated with common inherited variants in 227 candidate genes from ovarian cancer-related pathways including angiogenesis, inflammation, detoxification, glycosylation, one-carbon transfer, apoptosis, cell cycle regulation, and cellular senescence., Experimental Design: Blood samples were obtained from 325 women with invasive epithelial ovarian cancer diagnosed at the Mayo Clinic from 1999 to 2006. During a median follow-up of 3.8 years (range, 0.1-8.6 years), 157 deaths were observed. Germline DNA was analyzed at 1,416 single nucleotide polymorphisms (SNP). For all patients, and for 203 with serous subtype, we assessed the overall significance of each gene and pathway, and estimated risk of death via hazard ratios (HR) and 95% confidence intervals (CI), adjusting for known prognostic factors., Results: Variation within angiogenesis was most strongly associated with survival time overall (P = 0.03) and among patients with serous cancer (P = 0.05), particularly for EIF2B5 rs4912474 (all patients HR, 0.69; 95% CI, 0.54-0.89; P = 0.004), VEGFC rs17697305 (serous subtype HR, 2.29; 95% CI, 1.34-3.92; P = 0.003), and four SNPs in VHL. Variation within the inflammation pathway was borderline significant (all patients, P = 0.09), and SNPs in CCR3, IL1B, IL18, CCL2, and ALOX5 which correlated with survival time are worthy of follow-up., Conclusion: An extensive multiple-pathway assessment found evidence that inherited differences may play a role in outcome of ovarian cancer patients, particularly in genes within the angiogenesis and inflammation pathways. Our work supports efforts to target such mediators for therapeutic gain.

Purpose: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas., Experimental Design: A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-gamma enzyme-linked immunosorbent spot (ELIspot) assay., Results: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for > or =11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu(88-102) (p88)., Conclusion: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines.

Purpose: Women with advanced epithelial ovarian cancer are routinely treated with platinum-paclitaxel chemotherapy following cytoreductive surgery, yet only approximately 20% achieve long-term disease-free survival. We hypothesized that differences in gene expression before treatment could distinguish patients with short versus long time to recurrence after administration of platinum-paclitaxel combination chemotherapy., Experimental Design: To test this hypothesis, gene expression profiling of 79 primary surgically resected tumors from women with advanced-stage, high-grade epithelial ovarian cancer was done using cDNA microarrays containing 30,721 genes. Supervised learning algorithms were applied in an effort to develop a binary classifier that could discriminate women at risk for early (< or =21 months) versus late (>21 months) relapse after initial chemotherapy., Results: A 14-gene predictive model was developed using a set of training samples (n = 51) and subsequently tested using an independent set of test samples (n = 28). This model correctly predicted the outcome of 24 of the 28 test samples (86% accuracy) with 95% positive predictive value for early relapse., Conclusions: Predictive markers for early recurrence can be identified for platinum-paclitaxel combination chemotherapy in primary ovarian carcinoma. The proposed 14-gene model requires further validation.