Tumorgrafts as in vivo surrogates for women with ovarian cancer.

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.

Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen.

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15 amino acid epitopes (CEA.24, CEA.176/354, CEA.488, and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.

Functional folate receptor alpha is elevated in the blood of ovarian cancer patients.

BACKGROUND: Despite low incidence, ovarian cancer is the fifth leading cause of cancer deaths and it has the highest mortality rate of all gynecologic malignancies among US women. The mortality rate would be reduced with an early detection marker. The folate receptor alpha (FRalpha) is one logical choice for a biomarker because of its prevalent overexpression in ovarian cancer and its exclusive expression in only a few normal tissues. In prior work, it was observed that patients with ovarian cancer had elevated serum levels of a protein that bound to a FRalpha-specific monoclonal antibody relative to healthy individuals. However, it was not shown that the protein detected was intact functional FRalpha. In the current study, the goal was to determine whether ovarian cancer patients (n = 30) had elevated serum levels of a fully functional intact FRalpha compared to matched healthy controls (n = 30). METHODOLOGY/PRINCIPAL FINDINGS: FRalpha levels in serum were analyzed by two methods, immunoblotting analysis and a radiolabeled folic acid-based microfiltration binding assay. Using the immunoassay, we observed that levels of FRalpha were higher in serum of ovarian cancer patients as compared to controls. Similar results were also observed using the microfiltration binding assay, which showed that the circulating FRalpha is functional. Importantly, we also found that the levels of FRalpha were comparable between early and advanced stage patients. CONCLUSIONS: Our results demonstrate that ovarian cancer patients have elevated levels of functional intact FRalpha. These findings support the potential use of circulating FRalpha as a biomarker of early ovarian cancer.

An HLA-DR-degenerate epitope pool detects insulin-like growth factor binding protein 2-specific immunity in patients with cancer.

Recent studies have shown the importance of helper CD4 T cells in initiating and sustaining tumor-specific CD8 T-cell immunity. This has paved the way for identifying MHC class II epitopes that could be incorporated into class I-based vaccines. In this study, the goal was to identify an HLA-DR-degenerate epitope pool derived from insulin-like growth factor binding protein 2 (IGFBP-2). IGFBP-2, a regulator of insulin-like growth factor action, is overexpressed in the majority of breast and ovarian cancers. Using algorithms, we predicted 29 HLA-DR1-binding epitopes. Binding assays targeting 15 different HLA-DRs revealed that 10 epitopes were degenerate, binding to at least four different HLA-DR variants. An IFN-gamma enzyme-linked immunosorbent spot assay was used to assess immunity to these 10 epitopes in 48 patients with either breast or ovarian cancer and 18 controls. Elevated T-cell immunity in patients was detected in 4 of the 10 epitopes (IGFBP2.17, IGFBP2.22, IGFBP2.249, and IGFBP2.293). The cumulative T-cell frequency of these four epitopes was elevated in patients relative to controls. All four peptides are naturally processed and presented to CD4 T-cells. The degenerate pool of peptides covers nearly 80% of patients and may be useful for augmenting CD4 T-cell immunity in patients undergoing immunization.

T-cell immunity to the folate receptor alpha is prevalent in women with breast or ovarian cancer.

PURPOSE: Studies have demonstrated that the generation of immunity to tumor antigens is associated with improved prognosis for many cancers. A candidate antigen is the folate receptor alpha (FRalpha), which is overexpressed in breast and ovarian cancers. Our goal in this study was to attain a better understanding of the extent of endogenous FRalpha immunity. METHODS: Using a CD4+ T cell epitope prediction algorithm, we predicted promiscuous epitopes of FRalpha, and tested for immunity in 30 breast (n = 17) or ovarian (n = 13) cancer patients and 18 healthy donors using enzyme-linked immunospot analysis. RESULTS: Fourteen peptides were predicted, seven each from the carboxy- and amino-terminus halves of the protein. More than 70% of patients demonstrated immunity to at least one FRalpha peptide. Patients responded to an average of 3 +/- 0.5 peptides, whereas healthy donors responded to 1 +/- 0.4 peptides (P = .004). Five peptides were recognized by more than 25% of patients. Responses to three peptides were higher (P < .05) in patients than in healthy donors, suggesting augmented immunity. Compared with healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor (P = .03). There was no difference between each group in the responses to nonspecific (P = .2) and viral stimuli (P = .5). Lastly, patients demonstrated elevated levels of FRalpha antibodies consistent with a coordinated immune response. CONCLUSION: These findings demonstrate that the FRalpha is a target of the immune system in breast and ovarian cancer patients. Understanding which antigens are targeted by the immune system may be important for prognosis or immune-based therapies.