Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Voltage-Gated Sodium Channels as Potential Biomarkers and Therapeutic Targets for Epithelial Ovarian Cancer.

Abnormal ion channel expression distinguishes several types of carcinoma. Here, we explore the relationship between voltage-gated sodium channels (VGSC) and epithelial ovarian cancer (EOC). We find that EOC cell lines express most VGSC, but at lower levels than fallopian tube secretory epithelial cells (the cells of origin for most EOC) or control fibroblasts. Among patient tumor samples, lower SCN8A expression was associated with improved overall survival (OS) (median 111 vs. 52 months; HR 2.04 95% CI: 1.21-3.44; p = 0.007), while lower SCN1B expression was associated with poorer OS (median 45 vs. 56 months; HR 0.69 95% CI 0.54-0.87; p = 0.002). VGSC blockade using either anti-epileptic drugs or local anesthetics (LA) decreased the proliferation of cancer cells. LA increased cell line sensitivity to platinum and taxane chemotherapies. While lidocaine had similar additive effects with chemotherapy among EOC cells and fibroblasts, bupivacaine showed a more pronounced impact on EOC than fibroblasts when combined with either carboplatin (ΔAUC -37% vs. -16%, p = 0.003) or paclitaxel (ΔAUC -37% vs. -22%, p = 0.02). Together, these data suggest VGSC are prognostic biomarkers in EOC and may inform new targets for therapy.

Loss of Selenoprotein Iodothyronine Deiodinase 3 Expression Correlates with Progression of Complete Hydatidiform Mole to Gestational Trophoblastic Neoplasia.

To investigate if differences in imprinting at tropho-microRNA (miRNA) genomic clusters can distinguish between pre-gestational trophoblastic neoplasia cases (pre-GTN) and benign complete hydatidiform mole (CHM) cases at the time of initial uterine evacuation. miRNA sequencing was performed on frozen tissue from 39 CHM cases including 9 GTN cases. DIO3, DLK1, RTL1, and MEG 3 mRNA levels were assessed by qRT-PCR. Protein abundance was assessed by Western blot for DIO3, DLK1, and RTL1. qRT-PCR and Western blot were performed for selenoproteins and markers of oxidative stress. Immunohistochemistry (IHC) was performed for DIO3 on an independent validation set of clinical samples (n = 42) and compared to normal placenta controls across gestational ages. Relative expression of the 14q32 miRNA cluster was lower in pre-GTN cases. There were no differences in protein abundance of DLK1 or RTL1. Notably, there was lower protein expression of DIO3 in pre-GTN cases (5-fold, p < 0.03). There were no differences in mRNA levels of DIO3, DLK1, RTL1 or MEG 3. mRNA levels were higher in all CHM cases compared to normal placenta. IHC showed syncytiotrophoblast-specific DIO3 immunostaining in benign CHM cases and normal placenta, while pre-GTN cases of CHM lacked DIO3 expression. We describe two new biomarkers of pre-GTN CHM cases: decreased 14q32 miRNA expression and loss of DIO3 expression by IHC. Differences in imprinting between benign CHM and pre-GTN cases may provide insight into the fundamental development of CHM.

Distinct microRNA profiles for complete hydatidiform moles at risk of malignant progression.

BACKGROUND: MicroRNAs are small noncoding RNAs with important regulatory functions. Although well-studied in cancer, little is known about the role of microRNAs in premalignant disease. Complete hydatidiform moles are benign forms of gestational trophoblastic disease that progress to gestational trophoblastic neoplasia in up to 20% of cases; however, there is no well-established biomarker that can predict the development of gestational trophoblastic neoplasia. OBJECTIVE: This study aimed to investigate possible differences in microRNA expression between complete moles progressing to gestational trophoblastic neoplasia and those regressing after surgical evacuation. STUDY DESIGN: Total RNA was extracted from fresh frozen tissues from 39 complete moles collected at the time of uterine evacuation in Brazil. In the study, 39 cases achieved human chorionic gonadotropin normalization without further therapy, and 9 cases developed gestational trophoblastic neoplasia requiring chemotherapy. Total RNA was also extracted from 2 choriocarcinoma cell lines, JEG-3 and JAR, and an immortalized normal placenta cell line, 3A-subE. MicroRNA expression in all samples was quantified using microRNA sequencing. Hits from the sequencing data were validated using a quantitative probe-based assay. Significantly altered microRNAs were then subjected to target prediction and gene ontology analyses to search for alterations in key signaling pathways. Expression of potential microRNA targets was assessed by quantitative real-time polymerase chain reaction and western blot. Finally, potential prognostic protein biomarkers were validated in an independent set of formalin-fixed paraffin-embedded patient samples from the United States (15 complete moles progressing to gestational trophoblastic neoplasia and 12 that spontaneously regressed) using quantitative immunohistochemistry. RESULTS: In total, 462 microRNAs were identified in all samples at a threshold of <1 tag per million. MicroRNA sequencing revealed a distinct set of microRNAs associated with gestational trophoblastic neoplasia. Gene ontology analysis of the most altered transcripts showed that the leading pathway was related to response to ischemia (P<.001). Here, 2 of the top 3 most significantly altered microRNAs were mir-181b-5p (1.65-fold; adjusted P=.014) and mir-181d-5p (1.85-fold; adjusted P=.014), both of which have been shown to regulate expression of BCL2. By quantitative real-time polymerase chain reaction, BCL2 messenger RNA expression was significantly lower in the complete moles progressing to gestational trophoblastic neoplasia than the regressing complete moles (-4.69-fold; P=.018). Reduced expression of BCL2 was confirmed in tissue samples by western blot. Immunohistochemistry in the independent patient samples revealed significantly lower cytoplasmic expression of BCL2 in the villous trophoblasts from cases destined for progression to gestational trophoblastic neoplasia compared with those that regressed, both with respect to staining intensity (optic density 0.110±0.102 vs 0.212±0.036; P<.001) and to the percentage of positive cells (16%±28% vs 49.4%±28.05%; P=.003). CONCLUSION: Complete moles progressing to gestational trophoblastic neoplasia are associated with a distinct microRNA profile. miR-181 family members and BCL2 may be prognostic biomarkers for predicting gestational trophoblastic neoplasia risk.

Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian Cancer.

Purpose: A major challenge in platinum-based cancer therapy is the clinical management of chemoresistant tumors, which have a largely unknown pathogenesis at the level of epigenetic regulation.Experimental Design: We evaluated the potential of using global loss of 5-hydroxymethylcytosine (5-hmC) levels as a novel diagnostic and prognostic epigenetic marker to better assess platinum-based chemotherapy response and clinical outcome in high-grade serous tumors (HGSOC), the most common and deadliest subtype of ovarian cancer. Furthermore, we identified a targetable pathway to reverse these epigenetic changes, both genetically and pharmacologically.Results: This study shows that decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. In addition, global 5-hmC loss is associated with a decreased response to platinum-based chemotherapy, shorter time to relapse, and poor overall survival in patients newly diagnosed with HGSOC. Interestingly, the rescue of 5-hmC loss restores sensitivity to platinum chemotherapy in vitro and in vivo, decreases the percentage of tumor cells with cancer stem cell markers, and increases overall survival in an aggressive animal model of platinum-resistant disease.Conclusions: Consequently, a global analysis of patient 5-hmC levels should be included in future clinical trials, which use pretreatment with epigenetic adjuvants to elevate 5-hmC levels and improve the efficacy of current chemotherapies. Identifying prognostic epigenetic markers and altering chemotherapeutic regimens to incorporate DNMTi pretreatment in tumors with low 5-hmC levels could have important clinical implications for newly diagnosed HGSOC disease. Clin Cancer Res; 24(6); 1389-401. ©2017 AACR.

Type I interferons modulate methotrexate resistance in gestational trophoblastic neoplasia.

PROBLEM: Resistance to methotrexate is a leading clinical problem in gestational trophoblastic neoplasia (GTN), but there are limited laboratory models for this condition. METHOD OF STUDY: We created isogenic trophoblastic cell lines resistant to methotrexate and compared these to the parent cell lines using gene expression microarrays and qRT-PCR followed by mechanistic studies using recombinant cytokines, pathway inhibitors, and patient sera. RESULTS: Gene expression microarrays and focused analysis by qRT-PCR revealed methotrexate led to type I interferon upregulation, in particular interferon alpha 2 (IFNA2), and methotrexate resistance was associated with chronic low level increases in type I interferon expression. Recombinant IFNA2 imparted chemosensitive choriocarcinoma cells with partial resistance to methotrexate, while chemoresistant choriocarcinoma cells were uniquely sensitive to fludarabine, a STAT1 inhibitor. In pre-treatment patient sera, IFNA2 levels correlated with subsequent resistance to methotrexate chemotherapy. CONCLUSION: Methotrexate resistance is influenced by type I interferon signaling with prognostic and therapeutic implications for treating women with GTN.