Quantification of low-level human cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR.

BACKGROUND: Digital PCR (dPCR) can quantify cell-free viral DNA (DNAemia), a biomarker of active viral infection. To accelerate epidemiologic investigation into low-level viral reactivation in chronic disease, we have evaluated the performance of dPCR to detect cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia across platforms and blood matrices. METHODS: The droplet-based (BioRad) dPCR platform performance was compared to chip-based (BioMark), and assay validation followed dMIQE guidelines. CMV and EBV DNA reference materials were spiked into known negative plasma and serum samples. In addition, two independent cohorts of ovarian cancer patients were evaluated for viral DNAemia (n = 65 serum and 79 plasma samples). RESULTS: The limit of quantification (LOQ) was at or slightly above 100 copies/mL for both instruments: 105-135 copies/mL for droplet-based detection and 100 copies/mL for chip-based detection. DNAemia in serum had a slightly lower LOQ (105-110 copies/mL) compared to plasma (LOQ; 115-135 copies/mL). The variation (CV) coefficients for each assay and machine were less than 5 %. In patient samples, CVs ranged from 4.5 - 7.4 % and were similar for cell-free DNA derived from serum or plasma. There was good correlation between DNAemia measurements in patient samples across dPCR platforms (r > 0.90 for each assay and matrix). CONCLUSION: dPCR can quantify low-level herpes virus DNAemia with CVs below 8 %. Our results indicate that using serum-derived cell-free DNA and droplet-based dPCR is optimal for quantitating low-level viral DNAemia; however, plasma and chip-based approaches are acceptable alternatives and suitable for epidemiologic investigation.

Prevalence of active cytomegalovirus infection at diagnosis of ovarian cancer and during chemotherapy and subsequent changes in cognitive functioning.

PURPOSE: One of the most frequently reported effects of cancer and its treatments is cancer-related cognitive impairment (CRCI). Viral infections may affect inflammation and immune function and therefore may influence patient symptoms, including CRCI. The goal of this study was to describe the prevalence of cytomegalovirus (CMV) infections at diagnosis, during, and after chemotherapy in individuals with ovarian cancer and explore CMV infection at diagnosis with cancer-related cognitive impairment (CRCI) following chemotherapy. METHODS: We recruited adults newly diagnosed with ovarian, primary peritoneal or fallopian tube cancer at a single academic cancer center into two prospective studies. In Study 1 (N = 71), participants provided blood samples at diagnosis. In Study 2 (N = 18), participants provided blood samples and completed symptom surveys before, during and after front-line adjuvant chemotherapy. Serum CMV DNA levels were assessed using digital PCR; >100 copies/mL of serum was considered positive for active CMV infection (CMV+). CRCI was measured using the Functional Assessment of Cancer Therapy - Cognitive Function (FACT-Cog) questionnaire. Changes in FACT-Cog scores were compared by CMV status at diagnosis using t-tests at each time point. RESULTS: At diagnosis, 29.2% were CMV+ (28.2% in Study 1, 33.3% in Study 2). Following three cycles of chemotherapy (Study 2), CMV positivity rose to 60.0% and then back down to 31.3% after chemotherapy. We observed significant differences in CRCI following chemotherapy by CMV status at diagnosis. CONCLUSION: Our data suggest that active CMV infection is common among patients undergoing treatment for ovarian cancer and may contribute to symptoms of CRCI.

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of gynecologic cancer.

Advanced gynecologic cancers have historically lacked effective treatment options. Recently, immune checkpoint inhibitors (ICIs) have been approved by the US Food and Drug Administration for the treatment of cervical cancer and endometrial cancer, offering durable responses for some patients. In addition, many immunotherapy strategies are under investigation for the treatment of earlier stages of disease or in other gynecologic cancers, such as ovarian cancer and rare gynecologic tumors. While the integration of ICIs into the standard of care has improved outcomes for patients, their use requires a nuanced understanding of biomarker testing, treatment selection, patient selection, response evaluation and surveillance, and patient quality of life considerations, among other topics. To address this need for guidance, the Society for Immunotherapy of Cancer (SITC) convened a multidisciplinary panel of experts to develop a clinical practice guideline. The Expert Panel drew on the published literature as well as their own clinical experience to develop evidence- and consensus-based recommendations to provide guidance to cancer care professionals treating patients with gynecologic cancer.

Reverse Translation Identifies the Synergistic Role of Immune Checkpoint Blockade and IL15 to Enhance Immunotherapy of Ovarian Cancer.

Immune checkpoint blockade (ICB) has changed the standard of care for many patients with cancer, yet no ICB is approved for ovarian cancer. We hypothesized that maintenance therapy with an IL15 "superagonist" (N-803) and ICB in combination could induce potent immune activation in ovarian cancer. Using flow cytometry, cytometry by time of flight analysis, and cytotoxicity assays, we analyzed patient samples from women with advanced epithelial ovarian cancer treated with N-803 for indications of PD-1/PD-L1 upregulation with this treatment. In addition, ICB and N-803 were evaluated in preclinical studies to determine the functional impact of combination therapy on natural killer (NK) cells in vitro and in vivo. We observed that N-803 stimulated initial NK-cell expansion in patient samples; however, proliferation was not sustained beyond 2 weeks despite continued treatment. This result was reverse translated back to the laboratory to determine the functional relevance of this finding. The addition of ICB with an antibody-dependent cellular cytotoxicity IgG1 antibody against PD-L1 (avelumab) or an IgG4 antibody against PD-1 (pembrolizumab) enhanced N-803 induced NK-cell function in vitro. Using models of human ovarian cancer and NK-cell adoptive transfer in mice, we showed enhanced antitumor control with N-803 and ICB, as well as a combination effect that enhanced NK-cell persistence and expansion in vivo. This work suggests that PD-1/PD-L1 blockade combined with IL15 signaling may overcome resistance to cytokine therapy in ovarian cancer.

TEM8 Tri-specific Killer Engager binds both tumor and tumor stroma to specifically engage natural killer cell anti-tumor activity.

BACKGROUND: The tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting stromal cells, but current therapies targeting tumor stromal cells are constrained by modest efficacy and toxicities. TEM8 is a surface antigen selectively upregulated on tumor and tumor stromal cells, endothelial cells and fibroblasts that may be targeted with specific natural killer (NK) cell engagement. METHODS: A Tri-specific Killer Engager (TriKE) against TEM8-'cam1615TEM8'-was generated using a mammalian expression system. Its function on NK cells was assessed by evaluation of degranulation, inflammatory cytokine production, and killing against tumor and stroma cell lines in standard co-culture and spheroid assays. cam1615TEM8-mediated proliferation and STAT5 phosphorylation in NK cells was tested and compared with T cells by flow cytometry. NK cell proliferation, tumor infiltration, and tumor and tumor-endothelium killing by cam1615TEM8 and interleukin-15 (IL-15) were assessed in NOD scid gamma (NSG) mice. RESULTS: cam1615TEM8 selectively stimulates NK cell degranulation and inflammatory cytokine production against TEM8-expressing tumor and stromal cell lines. The increased activation translated to superior NK cell killing of TEM8-expressing tumor spheroids. cam1615TEM8 selectively stimulated NK cell but not T cell proliferation in vitro and enhanced NK cell proliferation, survival, and tumor infiltration in vivo. Finally, cam1615TEM8 stimulated NK cell killing of tumor and tumor endothelial cells in vivo. CONCLUSIONS: Our findings indicate that the cam1615TEM8 TriKE is a novel anti-tumor, anti-stroma, and anti-angiogenic cancer therapy for patients with solid tumors. This multifunctional molecule works by selectively targeting and activating NK cells by costimulation with IL-15, and then targeting that activity to TEM8+ tumor cells and TEM8+ tumor stroma.

Development of a Multiprotein Classifier for the Detection of Early Stage Ovarian Cancer.

BACKGROUND: Individual serum biomarkers are neither adequately sensitive nor specific for use in screening the general population for ovarian cancer. The purpose of this study was to develop a multiprotein classifier to detect the early stages of ovarian cancer, when it is most treatable. METHODS: The Olink Proseek Multiplex Oncology II panel was used to simultaneously quantify the expression levels of 92 cancer-related proteins in sera. RESULTS: In the discovery phase, we generated a multiprotein classifier that included CA125, HE4, ITGAV, and SEZ6L, based on an analysis of sera from 116 women with early stage ovarian cancer and 336 age-matched healthy women. CA125 alone achieved a sensitivity of 87.9% at a specificity of 95%, while the multiprotein classifier resulted in an increased sensitivity of 91.4%, while holding the specificity fixed at 95%. The performance of the multiprotein classifier was validated in a second cohort comprised of 192 women with early stage ovarian cancer and 467 age-matched healthy women. The sensitivity at 95% specificity increased from 74.5% (CA125 alone) to 79.2% with the multiprotein classifier. In addition, the multiprotein classifier had a sensitivity of 95.1% at 98% specificity for late stage ovarian cancer samples and correctly classified 80.5% of the benign samples using the 98% specificity cutpoint. CONCLUSIONS: The inclusion of the proteins HE4, ITGAV, and SEZ6L improved the sensitivity and specificity of CA125 alone for the detection of early stages of ovarian cancer in serum samples. Furthermore, we identified several proteins that may be novel biomarkers of early stage ovarian cancer.

What proportion of patients with stage 3 ovarian cancer are potentially cured following intraperitoneal chemotherapy? Analysis of the long term (≥10 years) survivors in NRG/GOG randomized clinical trials of intraperitoneal and intravenous chemotherapy in stage III ovarian cancer.

OBJECTIVE: Patients with advanced epithelial ovarian cancer (EOC) alive without progression at a landmark time-point of 10 years from diagnosis are likely cured. We report the proportion of patients with Stage III EOC who were long-term disease-free survivors (LTDFS≥10 years) following either intraperitoneal (IP) or intravenous (IV) chemotherapy as well as the predictors of LTDFS. METHODS: Data from 3 mature NRG/GOG trials (104, 114, 172) were analyzed and included demographics, clinicopathologic details, route of administration, and survival outcomes of patients living ≥10 years assessed according to the Kaplan-Meier method. Cox regression survival analysis was performed to evaluate independent prognostic predictors of LTDFS. RESULTS: Of 1174 patients randomized, 10-year overall survival (OS) was 26% (95% CI, 23-28%) and LTDFS ≥10 years was 18% (95% CI, 16-20%). Patients with LTDFS ≥10 years had a median age of 54.6 years (p < 0.001). Younger age (p < 0.001) was the only independent prognostic factor for LTDFS≥10 years on multivariate Cox analysis. CONCLUSIONS: Approximately 18% of patients were LTDFS ≥10 years. They form the tail end of the survival curve and are likely cured. Our results provide a comparative benchmark to evaluate the impact of PARP inhibitors in 1st line maintenance trials on survival outcomes.

Olaparib With or Without Cediranib Versus Platinum-Based Chemotherapy in Recurrent Platinum-Sensitive Ovarian Cancer (NRG-GY004): A Randomized, Open-Label, Phase III Trial.

PURPOSE: Platinum-based chemotherapy is the standard of care for platinum-sensitive ovarian cancer, but complications from repeated platinum therapy occur. We assessed the activity of two all-oral nonplatinum alternatives, olaparib or olaparib/cediranib, versus platinum-based chemotherapy. PATIENTS AND METHODS: NRG-GY004 is an open-label, randomized, phase III trial conducted in the United States and Canada. Eligible patients had high-grade serous or endometrioid platinum-sensitive ovarian cancer. Patients were randomly assigned 1:1:1 to platinum-based chemotherapy, olaparib, or olaparib/cediranib. The primary end point was progression-free survival (PFS) in the intention-to-treat population. Secondary end points included activity within germline BRCA-mutated or wild-type subgroups and patient-reported outcomes (PROs). RESULTS: Between February 04, 2016, and November 13, 2017, 565 eligible patients were randomly assigned. Median PFS was 10.3 (95% CI, 8.7 to 11.2), 8.2 (95% CI, 6.6 to 8.7), and 10.4 (95% CI, 8.5 to 12.5) months with chemotherapy, olaparib, and olaparib/cediranib, respectively. Olaparib/cediranib did not improve PFS versus chemotherapy (hazard ratio [HR] 0.86; 95% CI, 0.66 to 1.10; P = .077). In women with germline BRCA mutation, the PFS HR versus chemotherapy was 0.55 (95% CI, 0.32 to 0.94) for olaparib/cediranib and 0.63 (95% CI, 0.37 to 1.07) for olaparib. In women without a germline BRCA mutation, the PFS HR versus chemotherapy was 0.97 (95% CI, 0.73 to 1.30) for olaparib/cediranib and 1.41 (95% CI, 1.07 to 1.86) for olaparib. Hematologic adverse events occurred more commonly with chemotherapy; however, nonhematologic adverse events were higher with olaparib/cediranib. In 489 patients evaluable for PROs, patients receiving olaparib/cediranib scored on average 1.1 points worse on the NFOSI-DRS-P subscale (97.5% CI, -2.0 to -0.2, P = .0063) versus chemotherapy; no difference between olaparib and chemotherapy was observed. CONCLUSION: Combination olaparib/cediranib did not improve PFS compared with chemotherapy and resulted in reduced PROs. Notably, in patients with a germline BRCA mutation, both olaparib and olaparib/cediranib had significant clinical activity.

Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Chondroitin sulfate proteoglycan 4, a targetable oncoantigen that promotes ovarian cancer growth, invasion, cisplatin resistance and spheroid formation.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease encompassing several distinct molecular subtypes and clinical entities. Despite the initial success of surgical debulking and adjuvant chemotherapy, recurrence with chemotherapy resistant tumors is common in patients with EOC and leads to poor overall survival. The extensive genetic and phenotypic heterogeneity associated with ovarian cancers has hindered the identification of effective prognostic and predictive biomarkers in EOC patients. In the current studies, we identify a tumor cell surface oncoantigen, chondroitin sulfate proteoglycan 4 (CSPG4), as an independent risk factor for decreased survival of patients with EOC. Our results show that CSPG4 promotes EOC cell invasion, cisplatin resistance and spheroid formation in vitro and tumor expansion in vivo. Mechanistically, spheroid formation and tumor cell invasion are due to CSPG4-stimulated expression of the mesenchymal transcription factor ZEB1. Furthermore, we have developed a novel monoclonal anti-CSGP4 antibody against the juxtamembrane domain of the core protein that limits CSPG4-stimulated ZEB1 expression, tumor cell invasion and promotes EOC apoptosis within spheroid cultures. We therefore propose that CSPG4 expression drives phenotypic heterogeneity and malignant progression in EOC tumors. These studies further demonstrate that CSPG4 expression levels are a potential diagnostic biomarker in EOC and indicate that targeting cells which express this oncoantigen could limit recurrence and improve outcomes in patients with EOC.

A HER2 Tri-Specific NK Cell Engager Mediates Efficient Targeting of Human Ovarian Cancer.

Clinical studies validated antibodies directed against HER2, trastuzumab, and pertuzumab, as useful methodology to target breast cancer cases where HER2 is expressed. The hope was that HER2 targeting using these antibodies in ovarian cancer patients would prove useful as well, but clinical studies have shown lackluster results in this setting, indicating a need for a more comprehensive approach. Immunotherapy approaches stimulating the innate immune system show great promise, although enhancing natural killer (NK) function is not an established mainstream immunotherapy. This study focused on a new nanobody platform technology in which the bispecific antibody was altered to incorporate a cytokine. Herein we describe bioengineered CAM1615HER2 consisting of a camelid VHH antibody fragment recognizing CD16 and a single chain variable fragment (scFv) recognizing HER2 cross-linked by the human interleukin-15 (IL-15) cytokine. This tri-specific killer engager (TriKETM) showed in vitro prowess in its ability to kill ovarian cancer human cell lines. In addition, we demonstrated its efficacy in inducing potent anti-cancer effects in an in vivo xenograft model of human ovarian cancer engrafting both cancer cells and human NK cells. While previous approaches with trastuzumab and pertuzumab faltered in ovarian cancer, the hope is incorporating targeting and cytokine priming within the same molecule will enhance efficacy in this setting.

Radiotherapy Versus Inguinofemoral Lymphadenectomy as Treatment for Vulvar Cancer Patients With Micrometastases in the Sentinel Node: Results of GROINSS-V II.

PURPOSE: The Groningen International Study on Sentinel nodes in Vulvar cancer (GROINSS-V)-II investigated whether inguinofemoral radiotherapy is a safe alternative to inguinofemoral lymphadenectomy (IFL) in vulvar cancer patients with a metastatic sentinel node (SN). METHODS: GROINSS-V-II was a prospective multicenter phase-II single-arm treatment trial, including patients with early-stage vulvar cancer (diameter < 4 cm) without signs of lymph node involvement at imaging, who had primary surgical treatment (local excision with SN biopsy). Where the SN was involved (metastasis of any size), inguinofemoral radiotherapy was given (50 Gy). The primary end point was isolated groin recurrence rate at 24 months. Stopping rules were defined for the occurrence of groin recurrences. RESULTS: From December 2005 until October 2016, 1,535 eligible patients were registered. The SN showed metastasis in 322 (21.0%) patients. In June 2010, with 91 SN-positive patients included, the stopping rule was activated because the isolated groin recurrence rate in this group went above our predefined threshold. Among 10 patients with an isolated groin recurrence, nine had SN metastases > 2 mm and/or extracapsular spread. The protocol was amended so that those with SN macrometastases (> 2 mm) underwent standard of care (IFL), whereas patients with SN micrometastases (≤ 2 mm) continued to receive inguinofemoral radiotherapy. Among 160 patients with SN micrometastases, 126 received inguinofemoral radiotherapy, with an ipsilateral isolated groin recurrence rate at 2 years of 1.6%. Among 162 patients with SN macrometastases, the isolated groin recurrence rate at 2 years was 22% in those who underwent radiotherapy, and 6.9% in those who underwent IFL (P = .011). Treatment-related morbidity after radiotherapy was less frequent compared with IFL. CONCLUSION: Inguinofemoral radiotherapy is a safe alternative for IFL in patients with SN micrometastases, with minimal morbidity. For patients with SN macrometastasis, radiotherapy with a total dose of 50 Gy resulted in more isolated groin recurrences compared with IFL.

Sonosensitizer-Functionalized Graphene Nanoribbons for Adhesion Blocking and Sonodynamic Ablation of Ovarian Cancer Spheroids.

Advanced stage ovarian cancer is challenging to treat due to widespread seeding of tumor spheroids throughout the mesothelial lining of the peritoneal cavity. In this work, a therapeutic strategy using graphene nanoribbons (GNR) functionalized with 4-arm polyethylene glycol (PEG) and chlorin e6 (Ce6), a sonosensitizer, to target metastatic ovarian cancer spheroids is reported. GNR-PEG-Ce6 adsorbs onto the spheroids and disrupts their adhesion to extracellular matrix proteins or LP-9 mesothelial cells. Furthermore, for spheroids that do adhere, GNR-PEG-Ce6 delays spheroid disaggregation and spreading as well as mesothelial clearance, key metastatic processes following adhesion. Owing to the sonodynamic effects of Ce6 and its localized delivery via the biomaterial, GNR-PEG-Ce6 can kill ovarian cancer spheroids adhered to LP-9 cell monolayers when combined with mild ultrasound irradiation. The interaction with GNR-PEG-Ce6 also loosens cell-cell adhesions within the spheroids, rendering them more susceptible to treatment with the chemotherapeutic agents cisplatin and paclitaxel, which typically have difficulty in penetrating ovarian cancer spheroids. Thus, this material can facilitate effective chemotherapeutic and sonodynamic combination therapies. Finally, the adhesion inhibiting and sonodynamic effects of GNR-PEG-Ce6 are also validated with tumor spheroids derived from the ascites fluid of ovarian cancer patients, providing evidence of the translational potential of this biomaterial approach.

Differences in Family Planning and Fertility Among Female and Male Gynecologic Oncologists.

Background: The objective of the study was to compare family planning and infertility among female and male gynecologic oncologists in the United States Methods: This cross-sectional multiple choice survey was administered to the Society of Gynecologic Oncology gynecologic oncologists. The survey collected information on demographics and practice, family planning, and fertility and infertility experiences. Chi-square and Fisher's exact tests were used to compare experiences by gender. Results: Two hundred eighteen of 1243 (18%) members responded to the survey. The majority were women (71%), Caucasian (78%), and had been practicing fewer than 10 years (56%). One-third (32%) were 35+ years of age at the birth of their first child, and 67% delayed childbearing due to their career. Women were more likely than men to report career choice-influenced family planning. Just under half (44%) expressed current or past concerns about fertility, and this was more prevalent among women; 81% had sought infertility counseling. Among respondents who had fertility struggles, almost half (45%) reported their colleagues were unaware. Forty percent felt their fertility concerns affected work life, and 13% felt stigmatized for their fertility struggles. Conclusions: These findings suggest that a career in gynecologic oncology have an impact on family planning, often resulting in childbearing delays and infertility concerns, especially among women. Support for our colleagues struggling with infertility should be included in wellness initiatives.

Evaluation of the potential of Pap test fluid and cervical swabs to serve as clinical diagnostic biospecimens for the detection of ovarian cancer by mass spectrometry-based proteomics.

BACKGROUND: The purpose of this study was to determine whether the residual fixative from a liquid-based Pap test or a swab of the cervix contained proteins that were also found in the primary tumor of a woman with high grade serous ovarian cancer. This study is the first step in determining the feasibility of using the liquid-based Pap test or a cervical swab for the detection of ovarian cancer protein biomarkers. METHODS: Proteins were concentrated by acetone precipitation from the cell-free supernatant of the liquid-based Pap test fixative or eluted from the cervical swab. Protein was also extracted from the patient's tumor tissue. The protein samples were digested into peptides with trypsin, then the peptides were run on 2D-liquid chromatography mass spectrometry (2D-LCMS). The data was searched against a human protein database for the identification of peptides and proteins in each biospecimen. The proteins that were identified were classified for cellular localization and molecular function by bioinformatics integration. RESULTS: We identified almost 5000 proteins total in the three matched biospecimens. More than 2000 proteins were expressed in each of the three biospecimens, including several known ovarian cancer biomarkers such as CA125, HE4, and mesothelin. By Scaffold analysis of the protein Gene Ontology categories and functional analysis using PANTHER, the proteins were classified by cellular localization and molecular function, demonstrating that the Pap test fluid and cervical swab proteins are similar to each other, and also to the tumor extract. CONCLUSIONS: Our results suggest that Pap test fixatives and cervical swabs are a rich source of tumor-specific biomarkers for ovarian cancer, which could be developed as a test for ovarian cancer detection.

Cytomegalovirus and systemic inflammation at time of surgery is associated with worse outcomes in serous ovarian cancer.

OBJECTIVES: Cytomegalovirus (CMV) is a common infection that establishes latency in healthy people. CMV has been associated with alterations of the immune compartment leading to improved responses, while inflammation has been shown to adversely impact outcomes. We investigated whether CMV serostatus predicts outcomes in ovarian cancer in the presence or absence of inflammation. METHODS: A total of 106 patients with serous ovarian cancer from 2006 to 2009 were analyzed. CMV and systemic inflammation was measured using CMV immunoglobulin G (IgG) and C-reactive protein (CRP), respectively, in serum collected prior to cytoreduction. Patients were stratified by CMV IgG (non-reactive, reactive/borderline) and CRP (≤10, >10 mg/L) status. Overall survival (OS) and recurrence-free survival (RFS) were compared by group using log-rank tests and Cox proportional hazards regression models adjusting for age at surgery. RESULTS: Of 106 eligible patients, 40 (37.7%) were CMV+/CRP+, 24 (22.6%) CMV+/CRP-, 19 (17.9%) CMV-/CRP+, and 23 (21.7%) CMV-/CRP-. CRP+ had higher CA-125 levels (P = 0.05) and higher rates of suboptimal debulking (P = 0.03). There were no other significant differences in demographic, surgical, or pathologic factors between groups. CMV+/CRP+ patients median RFS and OS were 16.9 months (95% CI: 9.0-21.1) and 31.7 months (95% CI: 25.0-48.7), respectively, with a significantly worse RFS (aHR: 1.85, 95% CI: 1.05-3.24, P = 0.03) and OS (aHR: 2.12, 95% CI: 1.17-3.82, P = 0.01) compared to CMV-/CRP- (RFS = 31.2 months (95% CI: 16.0-56.4) and OS = 63.8 months (95% CI: 50.7-87.0)). CMV+/CRP- group displayed the longest OS (89.3 months). CONCLUSIONS: Previous exposure to CMV and high CRP at surgery portended worse RFS and OS compared to women who tested negative. The CMV+/CRP- group had the longest OS, indicating that CMV status alone, in the absence of inflammation, may be protective.

NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo.

We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKETM) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting.

Potent Cytolytic Activity and Specific IL15 Delivery in a Second-Generation Trispecific Killer Engager.

Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.

Immobilization rapidly selects for chemoresistant ovarian cancer cells with enhanced ability to enter dormancy.

Around 20-30% of ovarian cancer patients exhibit chemoresistance, but there are currently no methods to predict whether a patient will respond to chemotherapy. Here, we discovered that chemoresistant ovarian cancer cells exhibit enhanced survival in a quiescent state upon experiencing the stress of physical confinement. When immobilized in stiff silica gels, most ovarian cancer cells die within days, but surviving cells exhibit hallmarks of single-cell dormancy. Upon extraction from gels, the cells resume proliferation but demonstrate enhanced viability upon reimmobilization, indicating that initial immobilization selects for cells with a higher propensity to enter dormancy. RNA-seq analysis of the extracted cells shows they have signaling responses similar to cells surviving cisplatin treatment, and in comparison to chemoresistant patient cohorts, they share differentially expressed genes that are associated with platinum-resistance pathways. Furthermore, these extracted cells demonstrate greater resistance to cisplatin and paclitaxel, despite being proliferative. In contrast, serum starvation and hypoxia could not effectively select for chemoresistant cells upon removal of the environmental stress. These findings demonstrate that ovarian cancer chemoresistance and the ability to enter dormancy are linked, and immobilization rapidly distinguishes chemoresistant cells. This platform could be suitable for mechanistic studies, drug development, or as a clinical diagnostic tool.

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy.

Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.