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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.

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.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.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 (p0.001). Younger age (p0.001) was the only independent prognostic factor for LTDFS≥10 years on multivariate Cox analysis.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.

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.

Supplemental Table 1 and Supplemental Figures 1-5

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.

Supplemental Table S2

Animation of PCA for Figure 1

The best known ovarian cancer biomarker, CA125, is neither adequately sensitive nor specific for screening the general population. By using a combination of proteins for screening, it may be possible to increase the sensitivity and specificity over CA125 alone. In this study, we used Proseek Multiplex Oncology II plates to simultaneously measure the expression of 92 cancer-related proteins in serum using proximity extension assays. This technology combines the sensitivity of the PCR with the specificity of antibody-based detection methods, allowing multiplex biomarker detection and high-throughput quantification. We analyzed 1 μL of sera from each of 61 women with ovarian cancer and compared the values obtained with those from 88 age-matched healthy women. Principle component analysis and unsupervised hierarchical clustering separated the ovarian cancer patients from the healthy, with minimal misclassification. Data from the Proseek plates for CA125 levels exhibited a strong correlation with clinical values for CA125. We identified 52 proteins that differed significantly (P < 0.006) between ovarian cancer and healthy samples, several of which are novel serum biomarkers for ovarian cancer. In total, 40 proteins had an estimated area under the ROC curve of 0.70 or greater, suggesting their potential to serve as biomarkers for ovarian cancer. CA125 alone achieved a sensitivity of 93.4% at a specificity of 98%. By adding the Oncology II values for five proteins to CA125 in a multiprotein classifier, we increased the assay sensitivity to 98.4% at a specificity of 98%, thereby improving the sensitivity and specificity of CA125 alone.

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.

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.

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.

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 re-immobilization, 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.

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.

Simple Summary HER2 is a marker known to be over-expressed on breast cancer, rendering it one of the most useful solid tumor targets for antibody-based therapies. Despite expression on ovarian cancer, results targeting HER2 in this setting have been disappointing, thus requiring more aggressive approaches. Natural killer (NK) cells are known as principal mediators of cancer cell killing, but cancer cells find ways to deter them. We devised a tri-specific biological drug containing antibody fragments that simultaneously binds NK cells and cancer cells and at the same time delivers a natural cytokine signal that triggers robust NK cell expansion. In vitro studies show the drug augments NK cell killing of a number of HER2-positive human cell lines, while enhancing NK cell activation and proliferation. Studies in mice engrafted with human ovarian cancer showed the drug has anti-tumor efficacy, clearly demonstrating its ability to bolster NK cells in their ability to contain tumor cell growth. Abstract 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.

Objective Cervical cancer screening is often conducted in excess of current screening guidelines. The objective of this study was to evaluate the effect of an electronic health record (EHR) clinical decision support alert to decrease guideline-nonadherent cervical cancer screening beyond the age limits of screening or posthysterectomy. Materials and methods The proportion of guideline-nonadherent Pap tests in women younger than 21 years or older than 65 years or posthysterectomy were compared 4 months before and 3 months after implementation of an EHR clinical decision support alert warning providers that a Pap test is not indicated. Providers could cancel the Pap test or override the alert and place the order. Provider characteristics and Pap test indications were summarized by preintervention/postintervention period using descriptive statistics. The proportions of nonindicated Pap tests were compared by intervention period and provider characteristics using generalized estimating equation models. Results In women beyond the screening age limits or posthysterectomy, a total of 388 Pap tests were ordered before intervention, and 313 tests were ordered after intervention. Proportion of guideline-nonadherent tests was similar before (62%) and after intervention (63%); thus, implementation of the clinical decision support alert did not change the proportion of guideline-nonadherent Pap tests ordered (OR = 1.08, 95% CI = 0.77-1.52). It is notable that 52% of guideline-nonadherent tests were ordered by 11 providers. Even when controlling for providers who ordered more than 1 test during the study period, multivariate analysis showed that male providers were more likely to order guideline-nonadherent Pap tests (OR = 2.30, 95% CI = 1.36-3.89); no other differences by provider characteristics were observed. Conclusions An EHR clinical decision support alert does not decrease guideline-nonadherent cervical cancer screening. These data suggest efforts to optimize clinical decision support should be focused on other aspects of cervical cancer prevention.

Jacob Hutchins,1 Peter Argenta,2 Aaron Berg,1 Jason Habeck,1 Alexander Kaizer,3 Melissa A Geller21Department of Anesthesiology, University of Minnesota, Minneapolis, MN, USA; 2Department of Obstetrics, Gynecology and Women’s Health, Minneapolis, MN, USA; 3Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado, Aurora, CO, USAPurpose: To determine if a transversus abdominis plane (TAP) block with liposomal bupivacaine reduces total postoperative opioid use in the first 72 hrs following laparoscopic or robotic hysterectomy compared to port-site infiltration with 0.25% bupivacaine.Methods: Patients received either a true TAP block procedure with 266 mg liposomal bupivacaine and 50 mg of 0.25% bupivacaine and sham port infiltration or sham TAP block procedure with true port-site infiltration with 100–125 mg of 0.25% bupivacaine. All patients had a standardized, scheduled, non-opioid pain management plan. The primary outcome was total IV morphine equivalents used in the first 72 hrs following surgery. Secondary outcomes included assessment of postoperative pain over the study period and quality of recovery measures.Results: Patients undergoing TAP blockade required fewer total opioid equivalents during the observation period than patients allocated to infiltration (median 21 versus 25 mg IV Morphine equivalents, P=0.03). Opioid use was highest in the first 24 hrs after surgery, with less difference between the groups during days 2 and 3 postoperatively. There were 5 in the TAP group and 0 in the infiltration group were opioid free at 72 hrs. Those in the TAP group had improved quality of recovery (QoR15) with no change in overall benefit of analgesia score.Conclusion: TAP blockade reduced the requirement for opioid pain medication in the first 72 hrs after surgery, had more patients opioid free at 72 hrs, and improved patients’ quality of their recovery.Keywords: acute pain, regional pain, TAP block, liposome bupivacaine

Despite current guidelines recommending women with ovarian cancer receive genetic risk evaluation by a genetic counselor, utilization has historically been low. We sought to assess the feasibility and effectiveness of a week-long mobile Application for Genetic Information on Cancer (mAGIC) intervention aimed to persuade women with ovarian cancer to pursue genetic counseling.The mobile application intervention was based on the Fogg Behavior Model, and consisted of three parts: (1) identifying barriers, (2) developing motivators, and (3) providing triggers to action. The Health Belief Model was used to guide content development. We conducted a prospective, randomized, controlled pilot trial among 104 untested women with a history of epithelial ovarian, primary peritoneal or fallopian tube cancer with the primary objective of increasing uptake of cancer genetic counseling services.Utilization of cancer genetic counseling services improved in both study arms over historical controls, however there was no statistically significant difference between them (intervention: 54.5% versus control: 38.6%; p = 0.14). However, compared to controls, women randomized to the mAGIC intervention demonstrated greater knowledge of hereditary cancer (0-10 scale; 9.4 ± 1.0 vs. 7.1 ± 1.5; p 0.0001), which persisted for at least three months. Additionally, 96% of women in the intervention group reported they had talked with their family about genetic counseling compared to 77% in the control group (p = 0.01).The mAGIC intervention did not result in increased uptake of genetic counseling, however it provided significant secondary benefits, including increased participants' knowledge about hereditary ovarian cancer, self-efficacy, and their reported communication with family members. ClinicalTrials.gov Identifier: NCT02877862.

Objective Natural killer (NK) cells are lymphocytes well suited for adoptive immunotherapy. Attempts with adoptive NK cell immunotherapy against ovarian cancer have proven unsuccessful, with the main limitations including failure to expand and diminished effector function. We investigated if incubation of NK cells with interleukin (IL)-12, IL-15, and IL-18 for 16 h could produce cytokine-induced memory-like (CIML) NK cells capable of enhanced function against ovarian cancer. Methods NK cells were preactivated briefly with IL-12, IL-15, and IL-18, rested, then placed against ovarian cancer targets to assess phenotype and function via flow cytometry. Real-time NK-cell-mediated tumor-killing was evaluated. Using ascites cells and cell-free ascites fluid, NK cell proliferation and function within the immunosuppressive microenvironment was evaluated in vitro. Finally, CIML NK cells were injected intraperitoneal (IP) into an in vivo xenogeneic mouse model of ovarian cancer. Results CIML NK cells demonstrate enhanced cytokine (IFN-γ) production and NK-cell-mediated killing of ovarian cancer. NK cells treated overnight with cytokines led to robust activation characterized by temporal shedding of CD16, induction of CD25, and enhanced proliferation. CIML NK cells proliferate more with enhanced effector function compared to controls in an immunosuppressive microenvironment. Finally, human CIML NK cells exhibited potent antitumor effects within a xenogeneic mouse model of ovarian cancer. Conclusions CIML NK cells have enhanced functionality and persistence against ovarian cancer in vitro and in vivo, even when exposed to ascites fluid. These findings provide a strategy for NK cell-based immunotherapy to circumvent the immunosuppressive nature of ovarian cancer.

Objective To analyze clinical prognostic factors for survival after recurrence of high-grade, advanced-stage ovarian-peritoneal-tubal carcinoma and to develop a nomogram to predict individual survival after recurrence. Methods We retrospectively analyzed patients treated in multicenter Gynecologic Oncology Group protocols for stage III and IV ovarian-peritoneal-tubal carcinoma who underwent primary debulking surgery, received chemotherapy with paclitaxel and a platinum compound, and subsequently developed recurrence. Prognostic factors affecting survival were identified and used to develop a nomogram, which was both internally and externally validated. Results There were 4,739 patients included in this analysis, of whom, 84% had stage III and 16% had stage IV ovarian carcinoma. At a median follow-up of 88.8 months (95% CI 86.2-92.0 months), the vast majority of patients (89.4%) had died. The median survival after recurrence was 21.4 months (95% CI 20.5-21.9 months). Time to recurrence after initial chemotherapy, clear cell or mucinous histology, performance status, stage IV disease, and age were significant variables used to develop a nomogram for survival after recurrence, which had a concordance index of 0.67. The time to recurrence alone accounted for 85% of the prognostic information. Similar results were found for patients who underwent second look laparotomy and had a complete pathologic response or received intraperitoneal chemotherapy. Conclusion For individuals with advanced-stage ovarian carcinoma who recur after standard first-line therapy, estimated survivals after recurrence are closely related to the time to recurrence after chemotherapy and prognostic variables can be used to predict subsequent survival. Clinical trial registration ClinialTrials.gov, NCT00002568, NCT00837993, NCT00002717, NCT01074398, and NCT00011986.

BackgroundThe 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.MethodsA 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.Resultscam1615TEM8 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.ConclusionsOur 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.

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.

Simple Summary B7-H3 costimulatory ligand has sparked tremendous interest as a target for antibody-directed therapy of solid tumors. This is attributed to high expression of B7-H3 on the surface of tumor and low expression on normal tissues. Natural killer cells are known as key effectors of the innate immune system against cancer and are being explored clinically in a number of immunotherapy settings. Here, we describe a unique platform technology incorporating a cytokine as a bispecific antibody cross-linker to create a tri-specific NK cell engager, or TriKE, directed against the antigen B7-H3. The data shows this immunotherapeutic construct promotes specific natural killer cell expansion. It also induces antibody-dependent cellular cytotoxicity due to simultaneous recognition of both natural killer cells and B7-H3-expressing cancer cells. Finally, using a xenogeneic mouse model, the data shows that the TriKE induces better natural killer cell-mediated control of ovarian cancer. Taken together, the findings presented here indicate that a B7-H3-targeted TriKE has the potential to enhance natural killer cell immunotherapy in solid tumor settings and supports further development. Abstract 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.

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.

Purpose To determine the relationship between chemotherapy dose modification (dose adjustment or treatment delay), overall survival (OS) and progression-free survival (PFS) for women with advanced-stage epithelial ovarian carcinoma (EOC) and primary peritoneal carcinoma (PPC) who receive carboplatin and paclitaxel. Methods Women with stages III and IV EOC and PPC treated on the Gynecologic Oncology Group phase III trial, protocol 182, who completed eight cycles of carboplatin with paclitaxel were evaluated in this study. The patients were grouped per dose modification and use of granulocyte colony stimulating factor (G-CSF). The primary end point was OS; Hazard ratios (HR) for PFS and OS were calculated for patients who completed eight cycles of chemotherapy. Patients without dose modification were the referent group. All statistical analyses were performed using the R programming language and environment. Results A total of 738 patients were included in this study; 229 (31%) required dose modification, 509 did not. The two groups were well-balanced for demographic and prognostic factors. The adjusted hazard ratios (HR) for disease progression and death among dose-modified patients were: 1.43 (95% CI, 1.19–1.72, P Conclusion Dose-modified patients were at a higher risk of disease progression and death. The need for chemotherapy dose modification may identify patients at greater risk for adverse outcomes in advanced stage EOC and PPC.

OBJECTIVE: The aim of the study was to estimate the excess cost of guideline nonadherent cervical cancer screening in women beyond the recommended screening ages or posthysterectomy in a single healthcare system. MATERIALS AND METHODS: All Pap tests performed between September 1, 2012, and August 31, 2014, in women younger than 21 years, older than 65 years, or after hysterectomy, were coded as guideline adherent or nonadherent per the 2012 America Society of Colposcopy and Clinical Pathology guidelines. We assumed management of abnormal results per the 2013 America Society of Colposcopy and Clinical Pathology management guidelines. Costs were obtained from a literature review and Center for Medicare and Medicaid Services data and applied to nonadherent screening and subsequent diagnostic tests. RESULTS: During this period, 1,398 guideline nonadherent Pap tests were performed (257 in women 65 years, and 605 after hysterectomy), with 88 abnormal results: 35 (13.5%) in women younger than 21 years, 14 (2.6%) in women older than 65 years, and 39 (6.5%) in women after hysterectomy. The excess cost for initial screening, diagnostic tests, and follow-up was US $35,337 for 2 years in women younger than 21 years, US $54,378 for 5 years in women older than 65 years, and US $77,340 for 5 years in women after hysterectomy, resulting in a total excess cost of US $166,100 for 5 years. Of the 1,398 women who underwent guideline nonadherent screening, there were only 2 (0.1%) diagnoses of high-grade dysplasia (VaIN3). CONCLUSIONS: Guideline nonadherent cervical cancer screening in women beyond the recommended screening ages and posthysterectomy resulted in costs exceeding US $160,000 for screening, diagnostic tests, and follow-up with minimal improvement in detection of high-grade dysplasia.

A randomized controlled trial was conducted of a web-based intervention to improve advanced care planning in women with ovarian cancer. A secondary analysis of 35 randomized women focused on changes in distress and knowledge about ovarian cancer through distress monitoring and information tailored to patients' cognitive coping style (monitoring, blunting). Pre-/postresults indicated the Intervention group demonstrated lower distress (p = 0.06); blunting was associated with lower depression (p = 0.04); knowledge in both groups was unchanged. Women in the Intervention vs. Control group reported their family was less likely to be upset by cancer information (p = 0.0004). This intervention reduced distress while incorporating patient preferences.

To identify clinicopathologic factors associated with 10-year overall survival in epithelial ovarian cancer (EOC) and primary peritoneal cancer (PPC), and to develop a predictive model identifying long-term survivors.Demographic, surgical, and clinicopathologic data were abstracted from GOG 182 records. The association between clinical variables and long-term survival (LTS) (10years) was assessed using multivariable regression analysis. Bootstrap methods were used to develop predictive models from known prognostic clinical factors and predictive accuracy was quantified using optimism-adjusted area under the receiver operating characteristic curve (AUC).The analysis dataset included 3010 evaluable patients, of whom 195 survived greater than ten years. These patients were more likely to have better performance status, endometrioid histology, stage III (rather than stage IV) disease, absence of ascites, less extensive preoperative disease distribution, microscopic disease residual following cyoreduction (R0), and decreased complexity of surgery (p0.01). Multivariable regression analysis revealed that lower CA-125 levels, absence of ascites, stage, and R0 were significant independent predictors of LTS. A predictive model created using these variables had an AUC=0.729, which outperformed any of the individual predictors.The absence of ascites, a low CA-125, stage, and R0 at the time of cytoreduction are factors associated with LTS when controlling for other confounders. An extensively annotated clinicopathologic prediction model for LTS fell short of clinical utility suggesting that prognostic molecular profiles are needed to better predict which patients are likely to be long-term survivors.

Background The clinical assessment of circulating tumor cells (CTCs) as a blood-based biomarker is FDA-approved for use in breast, colorectal, and prostate cancers. The objective of this prospective clinical study was to determine whether pretreatment CTCs are a useful diagnostic biomarker in women with complex pelvic masses. Methods Whole blood was collected from 49 women with newly diagnosed pelvic masses. The presence of CTCs was compared between women with and without ovarian cancer histopathologic diagnosis after surgery using a Chi-squared test. Results CTCs were absent in those with benign disease (0/14), present in 17% (5/29) of patients with a histologic diagnosis of ovarian carcinoma, and present in 80% (4/5) of patients with ovarian metastases from other cancers (P = 0.001). All 5 women with ovarian cancer who had CTCs present presented stage III or IV of the disease (P = 0.13). Conclusions CTCs were more prevalent in patients with metastases to the ovary than in primary ovarian carcinomas.

Objectives The purpose of this study was to assess the prognostic significance of a simplified, clinically accessible classification system for endometrioid endometrial cancers combining Lynch syndrome screening and molecular risk stratification. Methods Tumors from NRG/GOG GOG210 were evaluated for mismatch repair defects (MSI, MMR IHC, and MLH1 methylation), POLE mutations , and loss of heterozygosity. TP53 was evaluated in a subset of cases. Tumors were assigned to four molecular classes. Relationships between molecular classes and clinicopathologic variables were assessed using contingency tests and Cox proportional methods. Results Molecular classification was successful for 982 tumors. Based on the NCI consensus MSI panel assessing MSI and loss of heterozygosity combined with POLE testing, 49% of tumors were classified copy number stable (CNS), 39% MMR deficient, 8% copy number altered (CNA) and 4% POLE mutant. Cancer-specific mortality occurred in 5% of patients with CNS tumors; 2.6% with POLE tumors; 7.6% with MMR deficient tumors and 19% with CNA tumors. The CNA group had worse progression-free (HR 2.31, 95%CI 1.53–3.49) and cancer-specific survival (HR 3.95; 95%CI 2.10–7.44). The POLE group had improved outcomes, but the differences were not statistically significant. CNA class remained significant for cancer-specific survival (HR 2.11; 95%CI 1.04–4.26) in multivariable analysis. The CNA molecular class was associated with TP53 mutation and expression status. Conclusions A simple molecular classification for endometrioid endometrial cancers that can be easily combined with Lynch syndrome screening provides important prognostic information. These findings support prospective clinical validation and further studies on the predictive value of a simplified molecular classification system.

Objectives: Identify if CMV reactivation allows for expansion of a special adaptive (natural killer) NK cell population with enhanced ability to kill ovarian cancer and determine if in a setting of increased inflammation (high CRP levels), this inflammatory response decreases the immunologic effect on tumor cells with an overall decrease in NK cell function and in cytokines necessary to activate immunogenic attacks. Methods: Serum (n=20 benign, 36 cancer) and ascites samples (n=18) collected in a prospective study at the time of surgery for HGSOC were evaluated for CMV and inflammation. CMV status and systemic inflammation were measured by immunoassay for CMV immunoglobulin G (IgG), dPCR for CMV DNA and C-reactive protein (CRP). Samples were categorized as reactive or non-reactive for CMV and positive or negative for CRP (≤10, >10 mg/L). Additionally, 32 HGOSC samples were measured for IL-6. Data were analyzed using SAS 9.4 (Cary, NC). P-values ≤ 0.05 were considered statistically significant. Flow cytometry was used to evaluate adaptive NK cells and assess cytotoxicity. Results: CMV reactivated patients had increased adaptive NK cells (NKG2C+CD57+) in serum and ascites compared to CMV non-reactived patients (p=0.043, p=0.077). In the serum, patients that were positive for CMV reactivation and were CRP negative had increased adaptive NK cells compared to patients that were CMV reactive and CRP positive. Elevated IL-6 levels in the serum and ascites correlated with CRP positive samples. There was no correlation between adaptive NK levels in blood and ascites in benign patient samples (r2=-0.03, p=0.46), however there was a positive correlation between adaptive NK levels in blood and ascites of ovarian cancer patient samples (r2=0.37 p=0.002). Adaptive NK cell levels correlate in CMV IgG positive cancer samples (r2=0.55, p=0.003), however CMV IgG negative samples did not correlate (r2=-0.04, p=0.45). Download : Download high-res image (45KB) Download : Download full-size image Conclusions: These findings suggest CMV reactivation increases the prevalence of adaptive NK cells in ovarian cancer patients.

Background Currently, there are no FDA approved screening tools for detecting early stage ovarian cancer in the general population. Development of a biomarker-based assay for early detection would significantly improve the survival of ovarian cancer patients. Methods We used a multiplex approach to identify protein biomarkers for detecting early stage ovarian cancer. This new technology (Proseek® Multiplex Oncology Plates) can simultaneously measure the expression of 92 proteins in serum based on a proximity extension assay. We analyzed serum samples from 81 women representing healthy, benign pathology, early, and advanced stage serous ovarian cancer patients. Results Principle component analysis and unsupervised hierarchical clustering separated patients into cancer versus non-cancer subgroups. Data from the Proseek® plate for CA125 levels exhibited a strong correlation with current clinical assays for CA125 (correlation coefficient of 0.89, 95% CI 0.83, 0.93). CA125 and HE4 were present at very low levels in healthy controls and benign cases, while higher levels were found in early stage cases, with highest levels found in the advanced stage cases. Overall, significant trends were observed for 38 of the 92 proteins (p

Objective Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. Methods NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays ±ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. Results ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. Conclusions ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK cell-based immunotherapeutic approaches for the treatment of ovarian cancer.

OBJECTIVE To estimate the proportion of guideline nonadherent Pap tests in women aged younger than 21 years and older than 65 years and posthysterectomy in a single large health system. Secondary objectives were to describe temporal trends and patient and health care provider characteristics associated with screening in these groups. METHODS A retrospective cross-sectional chart review was performed at Fairview Health Services and University of Minnesota Physicians. Reasons for testing and patient and health care provider information were collected. Tests were designated as indicated or nonindicated per the 2012 cervical cancer screening guidelines. Point estimates and descriptive statistics were calculated. Patient and health care provider characteristics were compared between indicated and nonindicated groups using χ and Wilcoxon rank-sum tests. RESULTS A total of 3,920 Pap tests were performed between September 9, 2012, and August 31, 2014. A total of 257 (51%; 95% confidence interval [CI] 46.1-54.9%) of tests in the younger than 21 years group, 536 (40%; 95% CI 37.7-43.1%) in the older than 65 years group, and 605 (29%; 95% CI 27.1-31.0%) in the posthysterectomy group were not indicated. White race in the older than 65 years group was the only patient characteristic associated with receipt of a nonindicated Pap test (P=.007). Health care provider characteristics associated with nonindicated Pap tests varied by screening group. Temporal trends showed a decrease in the proportion of nonindicated tests in the younger than 21 years group but an increase in the posthysterectomy group. CONCLUSION For women aged younger than 21 years and older than 65 years and posthysterectomy, 35% of Pap tests performed in our health system were not guideline-adherent. There were no patient or health care provider characteristics associated with guideline nonadherent screening across all groups.

Objectives The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells.

OBJECTIVE: To estimate whether a rapid recovery program would reduce length of stay among patients undergoing laparotomy on a gynecologic oncology service. METHODS: We conducted a prospective, randomized, controlled trial comparing an enhanced recovery after surgery protocol with routine postoperative care among women undergoing laparotomy on the gynecologic oncology service. Protocol elements included: preoperative counseling, regional anesthesia, intraoperative fluid restriction, and early postoperative ambulation and feeding. A sample size of 50 per group (N=100) was planned to achieve 80% power to detect a two-day difference in our primary outcome, length of hospital stay; secondary outcomes included: total daily narcotics used, time to postoperative milestones, and complications. RESULTS: A total of 112 women were enrolled between 2013 and 2015. Nine patients did not undergo laparotomy and were excluded, leaving 52 and 51 patients in the control and intervention groups, respectively. There was no difference in length of stay between the two groups (median 3.0 in both groups;P=.36). Enhanced recovery after surgery patients used less narcotics on day 0 (10.0 compared with 5.5 morphine equivalents in the control and intervention arms, respectively, P=.09) and day 2 (10.0 compared with 7.5 morphine equivalents, respectively; P=.05);however, there was no statistically significant difference between groups in any of the secondary outcomes. Post hoc analysis based on actual anesthesia received also failed to demonstrate a difference in time to discharge. CONCLUSION: When compared with usual care, introducing a formal enhanced recovery after surgery protocol did not significantly reduce length of stay. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT01705288.

Background Digital health provides solutions that capture patient-reported outcomes (PROs) and allows symptom monitoring and patient management. Digital therapeutics is the provision to patients of evidence-based therapeutic interventions through software applications aimed at prevention, monitoring, management, and treatment of symptoms and diseases or for treatment optimization. The digital health solutions collecting PROs address many unmet needs, including access to care and reassurance, increase in adherence and treatment efficacy, and decrease in hospitalizations. With current developments in oncology including increased availability of oral drugs and reduced availability of healthcare professionals, these solutions offer an innovative approach to optimize healthcare resource utilization. Design This scoping review clarifies the role and impact of the digital health solutions in oncology supportive care, with a view of the current segmentation according to their technical features (connection to sensors, PRO collection, remote monitoring, self-management in real time…), and identifies evidence from clinical studies published about their benefits and limitations and drivers and barriers to adoption. A qualitative summary is presented. Results Sixty-six studies were identified and included in the qualitative synthesis. Studies supported the use of 38 digital health solutions collecting ePROs and allowing remote monitoring, with benefits to patients regarding symptom reporting and management, reduction in symptom distress, decrease in unplanned hospitalizations and related costs and improved quality of life and survival. Among those 38 solutions 21 provided patient self-management with impactful symptom support, improvement of QoL, usefulness and reassurance. Principal challenges are in developing and implementing digital solutions to suit most patients, while ensuring patient compliance and adaptability for use in different healthcare systems and living environments. Conclusions There is growing evidence that digital health collecting ePROs provide benefits to patients related to clinical and health economic endpoints. These digital solutions can be integrated into routine supportive care in oncology practice to provide improved patient-centered care.

Introduction/Background Introduction: GROINSS-V II investigated whether radiotherapy is a safe alternative for inguinofemoral lymphadenectomy (IFL) in vulvar cancer patients with a metastatic sentinel node (SN). Methodology Methods: In GROINSS-V II, a prospective multicentre phase II trial, patients were included with early-stage squamous cell carcinoma (SCC) of the vulva (diameter Results From December 2005 until October 2016, 1552 eligible patients were registered. SN-metastasis occurred in 324/1552 (21%) patients. After 54 months of inclusion the stopping-rule for SN-positive patients was activated; interim analysis showed an increased risk for groin recurrence in case of SN-metastasis >2 mm and/or extranodal extension (ENE). After amendment of the protocol only patients with SN-micrometastasis ≤2 mm received radiotherapy, while those with SN metastasis >2 mm underwent IFL (with radiotherapy if >1 metastasis or ENE). Final analysis after ≥2 years of follow-up revealed six isolated groin recurrences in 157 patients with a SN-micrometastasis (3.8%). Four could not be considered radiotherapy failures: two developed recurrence in the contralateral (SN-negative) groin, two refused radiotherapy. Twenty-eight patients did not get radiotherapy (2 recurrences;7.1%). Among 129 patients who received radiotherapy to the groin(s) only two isolated groin recurrences were diagnosed (1.6%: 95%CI:0–3.8%). The combination of radiotherapy with SN was associated with minimal toxicity: 5/118(4.2%) grade 3 toxicity, no grade 4 or 5 toxicity. In 38/1222 SN-negative patients (3.1%: 95%CI:2.1–4.1%) isolated groin recurrences were diagnosed with clear protocol violations in 6 patients: incomplete treatment of the groin (n=3); primary tumor >4cm (n=1); not all SNs visualized on the lymphoscintigram removed (n=2). After exclusion of these protocol violations an isolated groin recurrence rate of 2.6% (95%CI:1.7–3.5%) was observed. Conclusion Radiotherapy to the groins is a safe alternative for IFL in patients with SN metastasis ≤2 mm, with minimal toxicity. We further established the safety of omitting IFL in patients with SCC of the vulva 2 mm, radiotherapy with a total dose of 50Gy was no safe alternative for IFL; dose escalation and/or chemoradiation should be investigated in these patients. Disclosure Funded by Dutch Cancer Society.

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.

The immunosuppressive tumor microenvironment limits the success of current immunotherapies. The host retains memory T cells specific for previous infections throughout the entire body that are capable of executing potent and immediate immunostimulatory functions. Here we show that virus-specific memory T cells extend their surveillance to mouse and human tumors. Reactivating these antiviral T cells can arrest growth of checkpoint blockade-resistant and poorly immunogenic tumors in mice after injecting adjuvant-free non-replicating viral peptides into tumors. Peptide mimics a viral reinfection event to memory CD8+ T cells, triggering antigen presentation and cytotoxic pathways within the tumor, activating dendritic cells and natural killer cells, and recruiting the adaptive immune system. Viral peptide treatment of ex vivo human tumors recapitulates immune activation gene expression profiles observed in mice. Lastly, peptide therapy renders resistant mouse tumors susceptible to PD-L1 blockade. Thus, re-stimulating known antiviral immunity may provide a unique therapeutic approach for cancer immunotherapy., The immunosuppressive tumor environment and the lack of functional anti-tumor immunity are major limiting factors in immunotherapy. Here the authors show that human and mouse tumors are infiltrated by virus-specific memory T cells, which can be harnessed by viral peptides to induce local and systemic anti-tumor immunity and synergize with checkpoint blockade.

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.