Jayanthi S. Lea

A STING-activating nanovaccine for cancer immunotherapy

Generation of tumor-specific T cells is critically important for cancer immunotherapy1,2. A major challenge in achieving a robust T cell response is the spatio-temporal orchestration of antigen cross-presentation in antigen presenting cells (APCs) with innate stimulation. Here we report a minimalist nanovaccine by a simple physical mixture of an antigen with a synthetic polymeric nanoparticle, PC7A NP, which generated a strong cytotoxic T cell response with low systemic cytokine expression. Mechanistically, PC7A NP achieved efficient cytosolic delivery of tumor antigens to APCs in draining lymph nodes leading to increased surface presentation while simultaneously activating type I interferon-stimulated genes. This effect was dependent on STING but not Toll-like receptor or MAVS pathway. Nanovaccine produced potent tumor growth inhibition in melanoma, colon cancer, and human papilloma virus-E6/E7 tumor models. Combination of PC7A nanovaccine with an anti-PD-1 antibody showed great synergy with 100% survival over 60 days in a TC-1 tumor model. Rechallenging of these tumor-free animals with TC-1 cells led to complete inhibition of tumor growth, suggesting generation of long-term antitumor memory. The STING-activating nanovaccine offers a simple, safe and robust strategy in boosting anti-tumor immunity for cancer immunotherapy.

Implications of EGFR inhibition in ovarian cancer cell proliferation

OBJECTIVES Epidermal Growth Factor Receptor (EGFR) is one of the four members of the Human Epidermal Receptor (HER) family and is over-expressed in multiple malignancies. EGFR over-expression in ovarian cancer has been associated with poor prognosis. Targeted inhibition of EGFR via its tyrosine kinase domain is a successful treatment in lung cancer. Our objective was to correlate EGFR over-expression and growth inhibition, by EGF receptor inhibitors, in ovarian cancer. MATERIALS AND METHODS HER expression in nine epithelial ovarian cancer cell lines and one lung cancer cell line was determined by Western blot analysis. EGFR phosphorylation sites were analyzed and DNA sequencing was performed. Cell proliferation assays were performed in the presence of the tyrosine kinase inhibitor, gefitinib, and the EGFR monoclonal antibody, cetuximab. Inhibitory concentrations of 50% of these therapies were determined and compared across all cell lines. The lung cancer cell line, HCC827, was used as a control. RESULTS Four of nine (44%) ovarian cancer cell lines and the control lung cancer cell line expressed EGFR. These same cell lines showed a common phosphorylated residue at position 992, while other residues were variably phosphorylated. All but one cell line expressed at least one HER family member. Mutational analysis of the ovarian cancer cell lines showed no mutations in EGFR exons 18-21. Cell proliferation assays using gefitinib and cetuximab showed minimal response in the ovarian cancer cell lines when compared to the control HCC827, but relative sensitivity compared to the one cell line that had no HER family expression. CONCLUSIONS Ovarian cancer cell lines show variable expression of activated EGFR. EGFR inhibition alone, in ovarian tumors that lack a tyrosine kinase mutation or over-express EGFR is unlikely to result in clinical response.

Oxygenation in cervical cancer and normal uterine cervix assessed using blood oxygenation level-dependent (BOLD) MRI at 3T†

Hypoxia is reported to be a biomarker for poor prognosis in cervical cancer. However, a practical noninvasive method is needed for the routine clinical evaluation of tumor hypoxia. This study examined the potential use of blood oxygenation level‐dependent (BOLD) contrast MRI as a noninvasive technique to assess tumor vascular oxygenation at 3T. Following Institutional Review Board‐approved informed consent and in compliance with the Health Insurance Portability and Accountability Act, successful results were achieved in nine patients with locally advanced cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) stage IIA to IVA] and three normal volunteers. In the first four patients, dynamic T2*‐weighted MRI was performed in the transaxial plane using a multi‐shot echo planar imaging sequence whilst patients breathed room air followed by oxygen (15 dm3/min). Later, a multi‐echo gradient echo examination was added to provide quantitative R2* measurements. The baseline T2*‐weighted signal intensity was quite stable, but increased to various extents in tumors on initiation of oxygen breathing. The signal in normal uterus increased significantly, whereas that in the iliacus muscle did not change. R2* responded significantly in healthy uterus, cervix and eight cervical tumors. This preliminary study demonstrates that BOLD MRI of cervical cancer at 3T is feasible. However, more patients must be evaluated and followed clinically before any prognostic value can be determined. Copyright

A phase I-II evaluation of veliparib (NSC #737664), topotecan, and filgrastim or pegfilgrastim in the treatment of persistent or recurrent carcinoma of the uterine cervix: an NRG Oncology/Gynecologic Oncology Group study.

Purpose The aim of this study was to evaluate the tolerability and efficacy of poly(ADP-ribose) polymerase (PARP) inhibition by veliparib during cytotoxic topotecan administration with filgrastim or pegfilgrastim neutrophil support in women with persistent or recurrent uterine cervix cancer. Experimental Design This phase I-II trial examined twice-daily oral veliparib (10 mg) given during once-daily intravenous topotecan (0.6 mg/m2) on days 1 to 5 of each treatment cycle. Cycles were repeated every 21 days until disease progression or until toxicity prohibited further therapy. Toxicity and objective response rate were primary endpoints. Results Twenty-seven women were enrolled. Frequently reported grade 3 or higher treatment-related toxicities were anemia (59%), thrombocytopenia (44%), leukopenia (22%), and neutropenia (19%). There were 2 partial responses (7% [90% confidence interval, 1%–22%]). Four patients had a disease progression date more than 6 months after the start of veliparib-topotecan therapy. Patients with low immunohistochemical expression (0–1+) of PARP-1 in their primary uterine cervix cancer were more likely to have a longer progression-free interval (hazard ratio, 0.25; P = 0.02) and survival (hazard ratio, 0.12; P = 0.005) after veliparib-topotecan therapy. Conclusions Clinical activity of a veliparib-topotecan combination was minimal in women with persistent or recurrent uterine cervix cancer. Women whose uterine cervix cancers express PARP-1 at low levels may benefit preferentially from PARP inhibitors combined with cytotoxic therapies, suggesting further study of PARP expression as an integral triage biomarker.

Secondary cytoreductive surgery for recurrent platinum‐sensitive ovarian cancer

To determine the risks and benefits of secondary cytoreductive surgery for recurrent platinum‐sensitive ovarian cancer.

LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment

Endometrial cancer is the most common gynecologic malignancy and the fourth most common malignancy in women. For most patients in whom the disease is confined to the uterus, treatment results in successful remission; however, there are no curative treatments for tumors that have progressed beyond the uterus. The serine/threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer, but the biological modes of action of LKB1 in this context remain incompletely understood. Here, we have shown that LKB1 suppresses tumor progression by altering gene expression in the tumor microenvironment. We determined that LKB1 inactivation results in abnormal, cell-autonomous production of the inflammatory cytokine chemokine (C-C motif) ligand 2 (CCL2) within tumors, which leads to increased recruitment of macrophages with prominent tumor-promoting activities. Inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression and increased survival. In human primary endometrial cancers, loss of LKB1 protein was strongly associated with increased CCL2 expression by tumor cells as well as increased macrophage density in the tumor microenvironment. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating potential avenues for therapeutic opportunities.

Secreted protein acidic and rich in cysteine as a regulator of murine ovarian cancer growth and chemosensitivity

OBJECTIVE Secreted protein acidic and rich in cysteine (SPARC) influences the growth of several solid tumors. Our objectives were to determine the effect of SPARC on the growth and response to cisplatin therapy of platinum-resistant ovarian cancer. STUDY DESIGN SPARC expression was determined in 4 platinum-resistant ovarian cancer cell lines. The effect of increasing SPARC on cell proliferation was determined in vitro. The effect of host-derived SPARC on tumor growth and response to therapy was determined in vivo using the murine ovarian cancer cell line, OSEID8, which was injected into the peritoneum of wild-type (WT) and SPARC-null (SP-/-) mice. RESULTS Forced expression of SPARC decreased growth of platinum-resistant ovarian cancer cell lines in vitro. In vivo, tumor growth was more aggressive in the absence of host-derived SPARC resulting in decreased survival compared with WT mice (P = .005). Cisplatin did not improve survival of WT mice. In contrast, cisplatin therapy resulted in a significant survival advantage (P = .0048) and decreased tumor volume (P = .02) in SP-/- animals. CONCLUSION We conclude that SPARC is an important extracellular matrix protein that regulates the growth and chemosensitivity of ovarian cancer. In general, SPARC appears to control tumor cell growth but also impede the efficacy of cisplatin therapy. Therefore, selective inhibition of SPARC may provide an attractive strategy for increasing the efficacy of therapy in platinum-resistant ovarian tumors.

Malignant ovarian germ cell tumor — Role of surgical staging and gonadal dysgenesis ☆

OBJECTIVE To evaluate the effect of comprehensive surgical staging and gonadal dysgenesis on the outcomes of patients with malignant ovarian germ cell tumor. METHODS We performed a retrospective review of patients with ovarian germ cell tumors who were treated at our institution between 1976 and 2012. RESULTS Malignant ovarian germ cell tumors (MOGCTs) were identified in 50 females. The median age was 24 years (range 13 to 49). Of all MOGCT patients, 42% had dysgerminoma, 20% immature teratoma, 16% endodermal sinus tumor, and 22% mixed germ cell tumor. Univariate analyses revealed that the lack of surgical staging (p=0.048) and endodermal sinus tumor (p=0.0085) were associated with disease recurrence, while age at diagnosis, ethnicity, and stage of the disease were not. Multivariate analyses revealed that the lack of surgical staging (p=0.029) and endodermal sinus tumor (p=0.016) were independently associated with disease recurrence. In addition, 7 patients (14%) had 46 XY karyotype, including 6 with pure dysgerminoma and 1 with mixed germ cell tumor. Five had Swyer syndrome and 2 had complete androgen insensitivity syndrome. Concurrent gonadoblastoma was found in 5 of the patients. No difference was found in the mean age at presentation, stage distribution, or recurrence rate for MOGCT patients with or without XY phenotype. CONCLUSIONS Comprehensive surgical staging was associated with a lower rate of recurrence. Fourteen percent of phenotypic females with MOGCT and 29% of those with dysgerminoma had XY karyotype. The clinical outcome of these patients is similar to that of MOGCT patients with XX karyotype.

Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development.

Objective: Loss of the fragile histidine triad (Fhit) protein has been documented in cervical cancer and dysplasia. The goal of this study was to confirm the utility of homozygous deletions, aberrant methylation, and immunohistochemical evaluations of FHIT as functionally relevant determinants of FHIT expression. Methods: We studied matched DNA, RNA, and protein from nine early-passage cervical cancer cell lines. DNA markers spanning FHIT were used to examine the extent of homozygous deletions for each cell line. 5′ CpG island methylation of FHIT was investigated by methylation-specific polymerase chain reaction (PCR) assays. FHIT transcripts were characterized by reverse transriptase (RT)-PCR. Western blot analysis and immunohistochemistry were performed to characterize Fhit protein expression. Results: Homozygous deletions were found in six of nine cervical cancer cell lines, but only had homozygous deletions involving an exon. All nine lines had both methylated and unmethylated alleles according to methylation-specific PCR. Loss of wild-type FHIT transcripts were found in five of nine lines. By western blot analysis, Fhit protein expression was lost in five of nine lines, producing an exact correlation with RT-PCR results. Immunohistochemical staining was concordant with Fhit protein expression by western blotting in eight of nine cell lines. Conclusion: A perfect correlation was found between FHIT mRNA expression and western blot analysis. Assays for Fhit protein expression and large FHIT homozygous deletions are representative biomarkers of Fhit expression. By contrast, the aberrant methylation assay is not concordant with FHIT gene expression, and we suggest caution in its use as a functionally relevant biomarker for cervical cancer.

Detection of phosphatidylserine-positive exosomes as a diagnostic marker for ovarian malignancies: a proof of concept study.

There are no suitable screening modalities for ovarian carcinomas (OC) and repeated imaging and CA-125 levels are often needed to triage equivocal ovarian masses. Definitive diagnosis of malignancy, however, can only be established by histologic confirmation. Thus, the ability to detect OC at early stages is low, and most cases are diagnosed as advanced disease. Since tumor cells expose phosphatidylserine (PS) on their plasma membrane, we predicted that tumors might secrete PS-positive exosomes into the bloodstream that could be a surrogate biomarker for cancer. To address this, we developed a highly stringent ELISA that detects picogram quantities of PS in patient plasma. Blinded plasma from 34 suspect ovarian cancer patients and 10 healthy subjects were analyzed for the presence of PS-expressing vesicles. The nonparametric Wilcoxon rank sum test showed the malignant group had significantly higher PS values than the benign group (median 0.237 vs. -0.027, p=0.0001) and the malignant and benign groups had significantly higher PS values than the healthy group (median 0.237 vs -0.158, p<0.0001 and -0.027 vs -0.158, p=0.0002, respectively). ROC analysis of the predictive accuracy of PS-expressing exosomes/vesicles in predicting malignant against normal, benign against normal and malignant against benign revealed AUCs of 1.0, 0.95 and 0.911, respectively. This study provides proof-of-concept data that supports the high diagnostic power of PS detection in the blood of women with suspect ovarian malignancies.