Angiogenesis-Enabled Human Ovarian Tumor Microenvironment-Chip Evaluates Pathophysiology of Platelets in Microcirculation.

The tumor microenvironment (TME) promotes angiogenesis for its growth through the recruitment of multiple cells and signaling mechanisms. For example, TME actively recruits and activates platelets from the microcirculation to facilitate metastasis, but platelets may simultaneously also support tumor angiogenesis. Here, to model this complex pathophysiology within the TME that involves a signaling triad of cancer cells, sprouting endothelial cells, and platelets, an angiogenesis-enabled tumor microenvironment chip (aTME-Chip) is presented. This platform recapitulates the convergence of physiology of angiogenesis and platelet function within the ovarian TME and describes the contribution of platelets in promoting angiogenesis within an ovarian TME. By including three distinct human ovarian cancer cell-types, the aTME-Chip quantitatively reveals the following outcomes-first, introduction of platelets significantly increases angiogenesis; second, the temporal dynamics of angiogenic signaling is dependent on cancer cell type; and finally, tumor-educated platelets either activated exogenously by cancer cells or derived clinically from a cancer patient accelerate tumor angiogenesis. Further, analysis of effluents available from aTME-Chip validate functional outcomes by revealing changes in cytokine expression and several angiogenic and metastatic signaling pathways due to platelets. Collectively, this tumor microphysiological system may be deployed to derive antiangiogenic targets combined with antiplatelet treatments to arrest cancer metastasis.

Efficacy and safety of autologous tumor-infiltrating lymphocytes in recurrent or refractory ovarian cancer, colorectal cancer, and pancreatic ductal adenocarcinoma.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy has shown efficacy in metastatic melanoma, non-small cell lung cancer, and other solid tumors. Our preclinical work demonstrated more robust CD8 predominant TIL production when agonistic anti-4-1BB and CD3 antibodies were used in early ex vivo TIL culture. METHODS: Patients with treatment-refractory metastatic colorectal (CRC), pancreatic (PDAC) and ovarian (OVCA) cancers were eligible. Lymphodepleting chemotherapy was followed by infusion of ex vivo expanded TIL, manufactured at MD Anderson Cancer Center with IL-2 and agonistic stimulation of CD3 and 4-1BB (urelumab). Patients received up to six doses of high-dose IL-2 after TIL infusion. Primary endpoint was evaluation of objective response rate at 12 weeks using Response Evaluation Criteria in Solid Tumors version 1.1 with secondary endpoints including disease control rate (DCR), duration of response, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: 17 patients underwent TIL harvest and 16 were treated on protocol (NCT03610490), including 8 CRC, 5 PDAC, and 3 OVCA patients. Median age was 57.5 (range 33-70) and 50% were females. Median number of lines of prior therapy was 2 (range 1-8). No responses were observed at 12 weeks. Ten subjects achieved at least one stable disease (SD) assessment for a DCR of 62.5% (95% CI 35.4% to 84.8%). Best response included prolonged SD in a patient with PDAC lasting 17 months. Median PFS and OS across cohorts were 2.53 months (95% CI 1.54 to 4.11) and 18.86 months (95% CI 4.86 to NR), respectively. Grade 3 or higher toxicities attributable to therapy were seen in 14 subjects (87.5%; 95% CI 61.7% to 98.4%). Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type and low expression of checkpoint markers. CONCLUSIONS: TIL manufactured with assistance of 4-1BB and CD3 agonism is feasible and treatment is associated with no new safety signals. While no responses were observed, a significant portion of patients achieved SD suggesting early/partial immunological effect. Further research is required to identify factors associated with resistance and functionally enhance T cells for a more effective therapy.

Classification of High-Grade Serous Ovarian Cancer Using Tumor Morphologic Characteristics.

Importance: Despite similar histologic appearance among high-grade serous ovarian cancers (HGSOCs), clinical observations suggest vast differences in gross appearance. There is currently no systematic framework by which to classify HGSOCs according to their gross morphologic characteristics. Objective: To develop and characterize a gross morphologic classification system for HGSOC. Design, Setting, and Participants: This cohort study included patients with suspected advanced-stage ovarian cancer who presented between April 1, 2013, and August 5, 2016, to the University of Texas MD Anderson Cancer Center, a large referral center. Patients underwent laparoscopic assessment of disease burden before treatment and received a histopathologic diagnosis of HGSOC. Researchers assigning morphologic subtype and performing molecular analyses were blinded to clinical outcomes. Data analysis was performed between April 2020 and November 2021. Exposures: Gross tumor morphologic characteristics. Main Outcomes and Measures: Clinical outcomes and multiomic profiles of representative tumor samples of type I or type II morphologic subtypes were compared. Results: Of 112 women (mean [SD] age 62.7 [9.7] years) included in the study, most patients (84% [94]) exhibited a predominant morphologic subtype and many (63% [71]) had a uniform morphologic subtype at all involved sites. Compared with those with uniform type I morphologic subtype, patients with uniform type II morphologic subtype were more likely to have a favorable Fagotti score (83% [19 of 23] vs 46% [22 of 48]; P = .004) and thus to be triaged to primary tumor reductive surgery. Similarly, patients with uniform type II morphologic subtype also had significantly higher mean (SD) estimated blood loss (639 [559; 95% CI, 391-887] mL vs 415 [527; 95% CI, 253-577] mL; P = .006) and longer mean (SD) operative time (408 [130; 95% CI, 350-466] minutes vs 333 [113; 95% CI, 298-367] minutes; P = .03) during tumor reductive surgery. Type I tumors had enrichment of epithelial-mesenchymal transition (false discovery rate [FDR] q-value, 3.10 × 10-24), hypoxia (FDR q-value, 1.52 × 10-5), and angiogenesis pathways (FDR q-value, 2.11 × 10-2), whereas type II tumors had enrichment of pathways related to MYC signaling (FDR q-value, 2.04 × 10-9) and cell cycle progression (FDR q-value, 1.10 × 10-5) by integrated proteomic and transcriptomic analysis. Abundances of metabolites and lipids also differed between the 2 morphologic subtypes. Conclusions and Relevance: This study identified 2 novel, gross morphologic subtypes of HGSOC, each with unique clinical features and molecular signatures. The findings may have implications for triaging patients to surgery or chemotherapy, identifying outcomes, and developing tailored therapeutic strategies.

Molecular Correlates of Venous Thromboembolism (VTE) in Ovarian Cancer.

BACKGROUND: The incidence of venous thromboembolism (VTE) in patients with ovarian cancer is higher than most solid tumors, ranging between 10-30%, and a diagnosis of VTE in this patient population is associated with worse oncologic outcomes. The tumor-specific molecular factors that may lead to the development of VTE are not well understood. OBJECTIVES: The aim of this study was to identify molecular features present in ovarian tumors of patients with VTE compared to those without. METHODS: We performed a multiplatform omics analysis incorporating RNA and DNA sequencing, quantitative proteomics, as well as immune cell profiling of high-grade serous ovarian carcinoma (HGSC) samples from a cohort of 32 patients with or without VTE. RESULTS: Pathway analyses revealed upregulation of both inflammatory and coagulation pathways in the VTE group. While DNA whole-exome sequencing failed to identify significant coding alterations between the groups, the results of an integrated proteomic and RNA sequencing analysis indicated that there is a relationship between VTE and the expression of platelet-derived growth factor subunit B (PDGFB) and extracellular proteins in tumor cells, namely collagens, that are correlated with the formation of thrombosis. CONCLUSIONS: In this comprehensive analysis of HGSC tumor tissues from patients with and without VTE, we identified markers unique to the VTE group that could contribute to development of thrombosis. Our findings provide additional insights into the molecular alterations underlying the development of VTE in ovarian cancer patients and invite further investigation into potential predictive biomarkers of VTE in ovarian cancer.

Erratum: Therapeutic Targeting of Follicular T Cells with Chimeric Antigen Receptor-Expressing Natural Killer Cells.

[This corrects the article DOI: 10.1016/j.xcrm.2020.100003.].

Therapeutic Targeting of Follicular T Cells with Chimeric Antigen Receptor-Expressing Natural Killer Cells.

Follicular helper T cells (TFH) are critical for vaccine and infection elicitation of long-lived humoral immunity, but exaggerated TFH responses can promote autoimmunity and other pathologies. It is unfortunate that no clinical interventions exist for the selective depletion of follicular T cells to alleviate these diseases. We engineered a chimeric antigen receptor (CAR) facilitating the specific targeting of cells with high expression levels of human programmed cell death protein 1 (PD-1), a cardinal feature of follicular T cells. CAR-expressing human natural killer (NK) cells robustly and discriminately eliminated PD-1high follicular human T cells in vitro and in a humanized mouse model of lupus-like disease while sparing B cells and other PD-1low T cell subsets, including regulatory T cells. These results establish a strategy for specific targeting of PD-1high T cells that can be advanced as a clinical tool for the selective depletion of pathogenic follicular T cells or other PD-1high target cells in certain disease states.

Molecular Analysis of Clinically Defined Subsets of High-Grade Serous Ovarian Cancer.

The diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups.

Perioperative trajectory of patient reported symptoms: a pilot study in gynecologic oncology patients.

OBJECTIVE: With the growing focus on patient-centered care, patient reported outcomes (PROs) are becoming an important component to clinical trials and quality metrics. The objective of this study was to pilot the collection of patient reported symptom burden in women undergoing surgery in a gynecologic oncology practice. METHODS: Perioperative patient reported symptom burden was measured for women undergoing laparotomy on the gynecologic oncology service at the University of Texas MD Anderson Cancer Center. Symptoms were assessed using the M.D. Anderson Symptom Inventory (MDASI-OC), a 27 item tool validated for use in patients with ovarian cancer. The MDASI-OC was administered as a preoperative baseline, daily while admitted to the hospital after surgery, twice a week on the first week after discharge and then weekly until 8 weeks postoperatively. RESULTS: 29 patients were evaluable. Seventy-five percent of patients had a diagnosis of ovarian cancer. Of those patients, half underwent a primary debulking surgery and the other half had neoadjuvant chemotherapy prior to interval cytoreductive surgery. In the postoperative inpatient setting, the five symptoms with the highest overall burden were fatigue, pain, abdominal pain, dry mouth and drowsiness. Longitudinal change of the top 5 symptoms during hospitalization did not show any significant difference between those who had neoadjuvant chemotherapy and those who did not. CONCLUSION: The collection of longitudinal PROs to assess symptom burden is feasible in patients undergoing gynecologic oncology surgery. Patient reported outcomes are a crucial component of patient-centered research and the longitudinal collection and analysis of symptom burden can allow for more meaningful comparisons of surgical technique and perioperative care.

Assessing maintenance of evaporative cooling systems in legionellosis outbreaks.

This study was designed to conduct systematic reviews of existing evaporative cooling system maintenance guidelines and of published Legionnaires' disease outbreaks to determine what, if any, maintenance practices were in place at the time of the disease outbreaks and then to contrast the reported practices with the published guidelines for evaporative cooling systems. For the first review, similarities in the reported recommendations were assessed; in the second review, any reported information about the state of the evaporative cooling system during the outbreak investigation was summarized. The systematic reviews yielded 38 current guidelines for evaporative cooling systems and 38 published outbreak investigations. The guidelines varied regarding the recommended type and dose of biocides, frequency of general inspections and total system maintenance, the preferred disinfection and cleaning procedures when testing a system for microbiological contamination, the type and frequency of testing procedures, and interpretation of test results. Overall, the maintenance guidelines did not contain sufficiently detailed procedures to prevent the problems that were observed in the outbreak investigations. These maintenance procedures included lack or improper use of a biocide; infrequent testing for microbiological contamination; improper use or maintenance of drift eliminators; and lack of a total system cleaning within 6 months of the outbreak for cooling systems that were either under continuous use, recently started up, or frequently switched on and off. This study suggests that more specific and standardized maintenance guidelines for the control of Legionella bacteria are needed and that these guidelines must be properly implemented to help reduce further Legionnaires' disease outbreaks associated with evaporative cooling systems.

Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7-hydroxystaurosporine (UCN-01).

Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7-hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.

RNA polymerase II stalled on a DNA template during transcription elongation is ubiquitinated and the ubiquitination facilitates displacement of the elongation complex.

When mammalian cells are exposed to cisplatin or ultraviolet irradiation, the RNA polymerase II (RNAP II) large subunit becomes ubiquitinated and is subsequently degraded via the proteasomal pathway. Using a DNA template immobilized on magnetic beads in an in vitro transcription reaction, we showed that a pause of the elongating RNAP II complex caused by nucleotide starvation induced the ubiquitination of the stalled RNAP II. The ubiquitinated RNAP II dissociated from the ternary complex when transcription was allowed to resume. The dissociated (free) RNAP II remained ubiquitinated. The proteasome inhibitor MG132 increased the accumulation of ubiquitinated free RNAP II but did not affect the amount of ubiquitinated, template-bound RNAP II, indicating that the ubiquitinated RNAP II was displaced from the template and then degraded by the proteasomes. Our work shows that the elongation complex that was stalled at the template by nucleotide starvation is targeted by the ubiquitin-conjugating system and that ubiquitination facilitates displacement of the stalled RNAP II from the template. Our findings together with the findings by others that DNA damaging agents induced the ubiquitination in mammalian cells that are nucleotide excision repair competent, suggest that the RNAP II ubiquitination may have a role in the regulation of transcription-coupled DNA repair.