Virally encoded interleukin-6 facilitates KSHV replication in monocytes and induction of dysfunctional macrophages.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi's sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Using scRNA-seq, we demonstrate that KSHV preferentially infects CD14+ monocytes, sustains viral lytic replication through the viral interleukin-6 (vIL-6), which activates STAT1 and 3, and induces an inflammatory gene expression program. To study the role of vIL-6 in monocytes upon KSHV infection, we generated recombinant KSHV with premature stop codon (vIL-6(-)) and its revertant viruses (vIL-6(+)). Infection of the recombinant viruses shows that both vIL-6(+) and vIL-6(-) KSHV infection induced indistinguishable host anti-viral response with STAT1 and 3 activations in monocytes; however, vIL-6(+), but not vIL-6(-), KSHV infection promoted the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL-6(+) KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL-6(-) KSHV infection or uninfected control. Notably, a viral nuclear long noncoding RNA (PAN RNA), which is required for sustaining KSHV gene expression, was substantially reduced in infected primary monocytes upon vIL-6(-) KSHV infection. These results highlight the critical role of vIL-6 in sustaining KSHV transcription in primary monocytes. Our findings also imply a clever strategy in which KSHV utilizes vIL-6 to secure its viral pool by expanding infected monocytes via differentiating into longer-lived dysfunctional macrophages. This mechanism may facilitate KSHV to escape from host immune surveillance and to support a lifelong infection.

KSHV vIL-6 enhances inflammatory responses by epigenetic reprogramming.

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and greater risks of other complications, including malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol(SH)-linked alkylation for the metabolic (SLAM) sequencing and Cleavage Under Target & Release Using Nuclease analysis (CUT&RUN), we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNA polymerase II with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-κB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitors, OTX015 and MZ1, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.

Use of population pharmacokinetic-pharmacodynamic modelling to inform antimalarial dose optimization in infants.

Infants bear a significant malaria burden but are usually excluded from participating in early dose optimization studies that inform dosing regimens of antimalarial therapy. Unlike older children, infants' exclusion from early-phase trials has resulted in limited evidence to guide accurate dosing of antimalarial treatment for uncomplicated malaria or malaria-preventive treatment in this vulnerable population. Subsequently, doses used in infants are often extrapolated from older children or adults, with the potential for under- or overdosing. Population pharmacokinetic-pharmacodynamic (PK-PD) modelling, a quantitative methodology that applies mathematical and statistical techniques, can aid the design of clinical studies in infants that collect sparse pharmacokinetic data as well as support the analysis of such data to derive optimized antimalarial dosing in this complex and at-risk yet understudied subpopulation. In this review, we reflect on what PK-PD modelling can do in programmatic settings of most malaria-endemic areas and how it can be used to inform antimalarial dose optimization for preventive and curative treatment of uncomplicated malaria in infants. We outline key developmental physiological changes that affect drug exposure in early life, the challenges of conducting dose optimization studies in infants, and examples of how PK-PD modelling has previously informed antimalarial dose optimization in this subgroup. Additionally, we discuss the limitations and gaps of PK-PD modelling when used for dose optimization in infants. To utilize modelling well, there is a need to generate useful, sparse, PK and PD data in this subpopulation to inform antimalarial optimal dosing in infancy.

Technical note: Minimizing CIED artifacts on a 0.35 T MRI-Linac using deep learning.

BACKGROUND: Artifacts from implantable cardioverter defibrillators (ICDs) are a challenge to magnetic resonance imaging (MRI)-guided radiotherapy (MRgRT). PURPOSE: This study tested an unsupervised generative adversarial network to mitigate ICD artifacts in balanced steady-state free precession (bSSFP) cine MRIs and improve image quality and tracking performance for MRgRT. METHODS: Fourteen healthy volunteers (Group A) were scanned on a 0.35 T MRI-Linac with and without an MR conditional ICD taped to their left pectoral to simulate an implanted ICD. bSSFP MRI data from 12 of the volunteers were used to train a CycleGAN model to reduce ICD artifacts. The data from the remaining two volunteers were used for testing. In addition, the dataset was reorganized three times using a Leave-One-Out scheme. Tracking metrics [Dice similarity coefficient (DSC), target registration error (TRE), and 95 percentile Hausdorff distance (95% HD)] were evaluated for whole-heart contours. Image quality metrics [normalized root mean square error (nRMSE), peak signal-to-noise ratio (PSNR), and multiscale structural similarity (MS-SSIM) scores] were evaluated. The technique was also tested qualitatively on three additional ICD datasets (Group B) including a patient with an implanted ICD. RESULTS: For the whole-heart contour with CycleGAN reconstruction: 1) Mean DSC rose from 0.910 to 0.935; 2) Mean TRE dropped from 4.488 to 2.877 mm; and 3) Mean 95% HD dropped from 10.236 to 7.700 mm. For the whole-body slice with CycleGAN reconstruction: 1) Mean nRMSE dropped from 0.644 to 0.420; 2) Mean MS-SSIM rose from 0.779 to 0.819; and 3) Mean PSNR rose from 18.744 to 22.368. The three Group B datasets evaluated qualitatively displayed a reduction in ICD artifacts in the heart. CONCLUSION: CycleGAN-generated reconstructions significantly improved both tracking and image quality metrics when used to mitigate artifacts from ICDs.

Screening for precancerous anal lesions linked to human papillomaviruses: French recommendations for clinical practice.

In France, about 2000 new cases of anal cancer are diagnosed annually. Squamous cell carcinoma is the most common histological type, mostly occurring secondary to persistent HPV16 infection. Invasive cancer is preceded by precancerous lesions. In addition to patients with a personal history of precancerous lesions and anal cancer, three groups are at very high risk of anal cancer: (i) men who have sex with men and are living with HIV, (ii) women with a history of high-grade squamous intraepithelial lesions (HSILs) or vulvar HPV cancer, and (iii) women who received a solid organ transplant more than 10 years ago. The purpose of screening is to detect HSILs so that they can be treated, thereby reducing the risk of progression to cancer. All patients with symptoms should undergo a proctological examination including standard anoscopy. For asymptomatic patients at risk, an initial HPV16 test makes it possible to target patients at risk of HSILs likely to progress to cancer. Anal cytology is a sensitive test for HSIL detection. Its sensitivity is greater than 80% and exceeds that of proctological examination with standard anoscopy. It is indicated in the event of a positive HPV16 test. In the presence of cytological abnormalities and/or lesions and a suspicion of dysplasia on clinical examination, high-resolution anoscopy is indicated. Performance is superior to that of proctological examination with standard anoscopy. However, this technique is not widely available, which limits its use. If high-resolution anoscopy is not possible, screening by a standard proctological examination is an alternative. There is a need to develop high-resolution anoscopy and triage tests and to evaluate screening strategies.

KSHV Topologically Associating Domains in Latent and Reactivated Viral Chromatin.

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a "bird cage" during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription. IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi's sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 bp resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral life cycle.

Clinical application of a template-guided automated planning routine.

PURPOSE: Determine the dosimetric quality and the planning time reduction when utilizing a template-based automated planning application. METHODS: A software application integrated through the treatment planning system application programing interface, QuickPlan, was developed to facilitate automated planning using configurable templates for contouring, knowledge-based planning structure matching, field design, and algorithm settings. Validations are performed at various levels of the planning procedure and assist in the evaluation of readiness of the CT image, structure set, and plan layout for automated planning. QuickPlan is evaluated dosimetrically against 22 hippocampal-avoidance whole brain radiotherapy patients. The required times to treatment plan generation are compared for the validations set as well as 10 prospective patients whose plans have been automated by QuickPlan. RESULTS: The generations of 22 automated treatment plans are compared against a manual replanning using an identical process, resulting in dosimetric differences of minor clinical significance. The target dose to 2% volume and homogeneity index result in significantly decreased values for automated plans, whereas other dose metric evaluations are nonsignificant. The time to generate the treatment plans is reduced for all automated plans with a median difference of 9' 50″ ± 4' 33″. CONCLUSIONS: Template-based automated planning allows for reduced treatment planning time with consistent optimization structure creation, treatment field creation, plan optimization, and dose calculation with similar dosimetric quality. This process has potential expansion to numerous disease sites.

Impact of Dolutegravir-Based Antiretroviral Therapy on Piperaquine Exposure following Dihydroartemisinin-Piperaquine Intermittent Preventive Treatment of Malaria in Pregnant Women Living with HIV.

Dihydroartemisinin-piperaquine, an artemisinin-based combination therapy, has been identified as a promising agent for intermittent preventive treatment of malaria in pregnancy. However, in pregnant women living with HIV (PLWH), efavirenz-based antiretroviral therapy (ART) significantly reduces the plasma exposure of piperaquine. In an open-label, nonrandomized, fixed-sequence, pharmacokinetic study, we compared piperaquine plasma concentrations in 13 pregnant women during a 3-day treatment course of dihydroartemisinin-piperaquine when coadministered with efavirenz-based versus dolutegravir-based ART in the second or third trimester of pregnancy. Piperaquine concentrations were measured over a 28-day period, while on efavirenz-based ART and after switching to dolutegravir-based ART. Noncompartmental analysis was performed, and geometric mean ratios (GMRs) and 90% confidence intervals (CIs) were generated to compare piperaquine pharmacokinetic parameters between these two treatment periods. Compared with efavirenz-based ART, coadministration of dihydroartemisinin-piperaquine and dolutegravir-based ART resulted in a 57% higher overall piperaquine exposure (area under the concentration-time curve from 0 to 672 h [AUC0-672 h]) (GMR, 1.57; 90% CI, 1.28 to 1.93). Piperaquine's day 7 concentrations were also 63% higher (GMR, 1.63; 90% CI, 1.29 to 2.11), while day 28 concentrations were nearly three times higher (GMR, 2.96; 90% CI, 2.25 to 4.07). However, the maximum piperaquine concentration (Cmax) remained similar (GMR, 1.09; 90% CI, 0.79 to 1.49). Dihydroartemisinin-piperaquine was well tolerated, with no medication-related serious adverse events observed in this small study. Compared with efavirenz-based ART, a known inducer of piperaquine metabolism, dolutegravir-based ART resulted in increased overall piperaquine exposure with pharmacokinetic parameter values that were similar to those published previously for pregnant and nonpregnant women. Our findings suggest that the efficacy of dihydroartemisinin-piperaquine will be retained in pregnant women on dolutegravir. (The study was registered on PACTR.samrc.ac.za [PACTR201910580840196].).

Effect of dihydroartemisinin/piperaquine for malaria intermittent preventive treatment on dolutegravir exposure in pregnant women living with HIV.

BACKGROUND: In sub-Saharan Africa, the burdens of malaria and HIV infections overlap. In settings with moderate-to-high malaria transmission intensity, pregnant women living with HIV (PLWH) require both ART and malaria intermittent preventive treatment (IPTp). Dihydroartemisinin/piperaquine has been identified as a promising alternative to sulfadoxine/pyrimethamine for IPTp. However, another antimalarial drug, artesunate/amodiaquine, similar to dihydroartemisinin/piperaquine, was previously shown to reduce dolutegravir exposure in non-pregnant adults. OBJECTIVES: To investigate the effect of dihydroartemisinin/piperaquine on dolutegravir plasma exposure in pregnant women on dolutegravir-based ART. METHODS: We conducted an open-label, non-randomized, fixed-sequence, pharmacokinetic study in PLWH in Malawi. Dolutegravir concentrations were measured over a 24 h period, before and after the recommended 3 day treatment dose of dihydroartemisinin/piperaquine in 12 pregnant women in their second or third trimester. Non-compartmental analysis was performed, and geometric mean ratios (GMRs) and 90% CIs were generated to compare dolutegravir pharmacokinetic parameters between the two treatment periods. RESULTS: Co-administration of dihydroartemisinin/piperaquine and dolutegravir increased dolutegravir's overall exposure (AUC0-24) and Cmax by 30% (GMR 1.30; 90% CI 1.11-1.52) and 31% (GMR 1.31; 90% CI 1.13-1.51), respectively. The dolutegravir trough (C24) concentration increased by 42% (GMR 1.42; 90% CI 1.09-1.85). The combined treatments were well tolerated with no serious adverse events observed. CONCLUSIONS: Dihydroartemisinin/piperaquine may be administered with dolutegravir-based ART in pregnant women as the modest increase in dolutegravir exposure, similar to pharmacokinetic parameter values published previously, ensures its efficacy without any clinically significant adverse events observed in this small study.

Cerebral Regional Tissue Oxygenation as Surrogate for Blood Loss in Nonhuman Primate Models of Shock.

INTRODUCTION: Hemorrhage is the leading cause of preventable death, with a majority of mortalities in the prehospital setting. Current hemorrhage resuscitation guidelines cannot predict the critical point of intervention to activate massive transfusion (MT) and prevent cardiovascular decompensation. We hypothesized that cerebral regional tissue oxygenation (CrSO2) would indicate MT need in nonhuman primate models of hemorrhagic shock. METHODS: Nineteen anesthetized male rhesus macaques underwent hemorrhage via a volume-targeted (VT) or pressure-targeted (PT) method. VT animals were monitored for 30 min following 30% blood volume hemorrhage. PT animals were hemorrhaged to mean arterial pressure (MAP) of 20 mmHg and maintained for at least 60 min until decompensation. Statistics for MAP, heart rate (HR), end tidal carbon dioxide (EtCO2), and CrSO2 were analyzed via one- or two-way repeated-measures analysis of variance, Pearson's R, and receiver-operator curve. A P < 0.05 is considered significant. RESULTS: Following initial hemorrhage (S0), there were no significant differences between groups. After cessation of hemorrhage in the VT group, MAP and EtCO2 returned to baseline while CrSO2 plateaued. The PT group maintained model-defined low MAP, suppressing EtCO2, and significantly decreased CrSO2 compared to the VT group by S25. Linear regression of CrSO2versus shed blood volume demonstrated R2 = 0.7539. CrSO2 of 47% was able to detect >40% blood loss with an area under the curve of 0.9834 at 92.3% (66.7%-99.6%) sensitivity and 95.5% (84.9%-99.2%) specificity. CONCLUSIONS: Regardless of hemorrhage modality and compensatory response, CrSO2 correlated strongly with shed blood volume. Analysis demonstrated that CrSO2 values below 49% indicate Advanced Trauma Life Support class IV shock (blood loss>40%). CrSO2 at the point of care may help indicate MT need earlier and more accurately than traditional markers.

Early doxorubicin cardiotoxicity in Malawian children admitted to Queen Elizabeth Central Hospital, Malawi.

BACKGROUND: Doxorubicin is known to cause chemotherapy-induced cardiotoxicity. In resource-poor settings, monitoring for cardiotoxicity is not routinely done, and its incidence is unknown. The aim of this study was to determine the proportion of children who developed doxorubicin-induced cardiotoxicity within 1 year of having received treatment at paediatric oncology ward. METHODS: Children aged 3 months to 18 years with cancer were prospectively enrolled and followed up between January 2016 to June 2019. Transthoracic echocardiogram was done at baseline, 1 month, 6 months and a year after completion of therapy. Cardiotoxicity was defined as a decline in left ventricular ejection fraction (LVEF) of ≥10% to a final value of <50%. An overall incidence risk of developing cardiotoxicity was estimated. A one-way analysis of variance (ANOVA) was conducted to compare baseline LVEF with follow-up measurements. FINDINGS: Ninety-one children were enrolled, 74% (68/91) were male, and 67% (62/91) were aged 5 months to 14 years. Most patients received a doxorubicin cumulative dose between 100 and 200 mg/m2 and no cardiotoxicity was observed during the study period. However, of 77 children with at least one follow up, five children 6.54% (95% CI: 2.1-14.5) experienced LVEF reduction of >10%, though not to a final value of <50%. No deterioration of systolic function was found among 20 children who completed follow-up (F = 2.43, p-value = .07). INTERPRETATION: In this cohort of patients, most received a low cumulative doxorubicin dose and only 22% were available for evaluation at study end; no cardiotoxic events associated with doxorubicin administration were observed after 12 months.

Phase I/II Trial of Electrophysiology-Guided Noninvasive Cardiac Radioablation for Ventricular Tachycardia.

BACKGROUND: Case studies have suggested the efficacy of catheter-free, electrophysiology-guided noninvasive cardiac radioablation for ventricular tachycardia (VT) using stereotactic body radiation therapy, although prospective data are lacking. METHODS: We conducted a prospective phase I/II trial of noninvasive cardiac radioablation in adults with treatment-refractory episodes of VT or cardiomyopathy related to premature ventricular contractions (PVCs). Arrhythmogenic scar regions were targeted by combining noninvasive anatomic and electric cardiac imaging with a standard stereotactic body radiation therapy workflow followed by delivery of a single fraction of 25 Gy to the target. The primary safety end point was treatment-related serious adverse events in the first 90 days. The primary efficacy end point was any reduction in VT episodes (tracked by indwelling implantable cardioverter defibrillators) or any reduction in PVC burden (as measured by a 24-hour Holter monitor) comparing the 6 months before and after treatment (with a 6-week blanking window after treatment). Health-related quality of life was assessed using the Short Form-36 questionnaire. RESULTS: Nineteen patients were enrolled (17 for VT, 2 for PVC cardiomyopathy). Median noninvasive ablation time was 15.3 minutes (range, 5.4-32.3). In the first 90 days, 2/19 patients (10.5%) developed a treatment-related serious adverse event. The median number of VT episodes was reduced from 119 (range, 4-292) to 3 (range, 0-31; P<0.001). Reduction was observed for both implantable cardioverter defibrillator shocks and antitachycardia pacing. VT episodes or PVC burden were reduced in 17/18 evaluable patients (94%). The frequency of VT episodes or PVC burden was reduced by 75% in 89% of patients. Overall survival was 89% at 6 months and 72% at 12 months. Use of dual antiarrhythmic medications decreased from 59% to 12% ( P=0.008). Quality of life improved in 5 of 9 Short Form-36 domains at 6 months. CONCLUSIONS: Noninvasive electrophysiology-guided cardiac radioablation is associated with markedly reduced ventricular arrhythmia burden with modest short-term risks, reduction in antiarrhythmic drug use, and improvement in quality of life. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/ . Unique identifier: NCT02919618.

Noninvasive Cardiac Radiation for Ablation of Ventricular Tachycardia.

BACKGROUND: Recent advances have enabled noninvasive mapping of cardiac arrhythmias with electrocardiographic imaging and noninvasive delivery of precise ablative radiation with stereotactic body radiation therapy (SBRT). We combined these techniques to perform catheter-free, electrophysiology-guided, noninvasive cardiac radioablation for ventricular tachycardia. METHODS: We targeted arrhythmogenic scar regions by combining anatomical imaging with noninvasive electrocardiographic imaging during ventricular tachycardia that was induced by means of an implantable cardioverter-defibrillator (ICD). SBRT simulation, planning, and treatments were performed with the use of standard techniques. Patients were treated with a single fraction of 25 Gy while awake. Efficacy was assessed by counting episodes of ventricular tachycardia, as recorded by ICDs. Safety was assessed by means of serial cardiac and thoracic imaging. RESULTS: From April through November 2015, five patients with high-risk, refractory ventricular tachycardia underwent treatment. The mean noninvasive ablation time was 14 minutes (range, 11 to 18). During the 3 months before treatment, the patients had a combined history of 6577 episodes of ventricular tachycardia. During a 6-week postablation "blanking period" (when arrhythmias may occur owing to postablation inflammation), there were 680 episodes of ventricular tachycardia. After the 6-week blanking period, there were 4 episodes of ventricular tachycardia over the next 46 patient-months, for a reduction from baseline of 99.9%. A reduction in episodes of ventricular tachycardia occurred in all five patients. The mean left ventricular ejection fraction did not decrease with treatment. At 3 months, adjacent lung showed opacities consistent with mild inflammatory changes, which had resolved by 1 year. CONCLUSIONS: In five patients with refractory ventricular tachycardia, noninvasive treatment with electrophysiology-guided cardiac radioablation markedly reduced the burden of ventricular tachycardia. (Funded by Barnes-Jewish Hospital Foundation and others.).

Short-term organoid culture for drug sensitivity testing of high-grade serous carcinoma.

OBJECTIVE: Cancer patient-derived organoids (PDOs) grow as three dimensional (3D) structures in the presence of extracellular matrix and have been found to represent the original tumor's genetic complexity. In addition, PDOs can be grown and subjected to drug sensitivity testing in a shorter time course and with lesser expense than patient-derived xenograft models. Many patients with recurrent ovarian cancer develop malignant effusions that become refractory to chemotherapy. Since these same patients often present for palliative aspiration of ascites or pleural effusions, there is a potential opportunity to obtain tumor specimens in the form of multicellular spheroids (MCS) present in malignant effusion fluids. Our objective was to develop a short duration culture of MCS from ovarian cancer malignant effusions in conditions selected to support organoid growth and use them as a platform for empirical drug sensitivity testing. METHODS: In this study, malignant effusion specimens were collected from patients with high-grade serous ovarian carcinoma (HGSOC). MCS were recovered and subjected to culture conditions designed to support organoid growth. In a subset of specimens, RNA-sequencing was performed at two time points during the short-term culture to determine changes in transcriptome in response to culture conditions. Organoid induction was also characterized in these specimens using Ki67 staining and histologic analysis. Drug sensitivity testing was performed on all specimens. RESULTS: Our model describes organoids formed within days of primary culture, which can recapitulate the histological features of malignant ascites fluid and can be expanded for at least 6 days. RNA-seq analysis of four patient specimens showed that within 6 days of culture, there was significant up-regulation of genes related to cellular proliferation, epithelial-mesenchymal transition, and KRAS signaling pathways. Drug sensitivity testing identified several agents with therapeutic potential. CONCLUSIONS: Short duration organoid culture of MCS from HGSOC malignant effusions can be used as a platform for empiric drug sensitivity testing. These ex vivo models may be helpful in screening new or existing therapeutic agents prior to individualized treatment options.

Immunotherapy and Radiation Therapy for Non-Small Cell Lung Cancer-A Stimulating Partnership.

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer and the leading cause of cancer-related death. Although durable local control rates are high after surgical resection or definitive radiotherapy for early-stage disease, a substantial proportion of these patients eventually experience regional and/or distant failure and succumb to their metastatic disease. The discovery of immunotherapeutics and targeted biologics has revolutionized the treatment of locally advanced and metastatic disease, improving progression-free and overall survival when incorporated with the current standards of care. Notably, post-hoc analyses and early clinical trials provide a growing body of evidence to support a synergistic effect between radiation and immunotherapy for the treatment of NSCLC from early-stage to metastatic disease. Radiotherapy appears to be capable of not only potentiating the effect of immunotherapy in targeted lesions, but also eliciting an antitumor response in distant lesions without any direct exposure to radiation. This review explores the biologic basis of immunotherapy, targeted biologics, and radiotherapy as well as the preclinical and clinical data that support the combined use of radioimmunotherapy for early-stage, locally advanced, and metastatic NSCLC.

A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials.

In order to develop a practical model of breast cancer, with in vitro and syngeneic, immune-intact, in vivo growth capacity, we established a primary cell line derived from a mammary carcinoma in the transgenic FVB/N-Tg(MMTV-ErbB2*)NDL2-5Mul mouse, referred to as "NDLUCD". The cell line is adapted to standard cell culture and can be transplanted into syngeneic FVB/N mice. The line maintains a stable phenotype over multiple in vitro passages and rounds of in vivo transplantation. NDLUCD tumors in FVB/N mice exhibit high expression of ErbB2 and ErbB3 and signaling molecules downstream of ErbB2. The syngeneic transplant tumors elicit an immune reaction in the adjacent stroma, detected and characterized using histology, immunophenotyping, and gene expression. NDLUCD cells also express PD-L1 in vivo and in vitro, and in vivo transplants are reactive to anti-immune checkpoint therapy with responses conducive to immunotherapy studies. This new NDLUCD cell line model is a practical alternative to the more commonly used 4T1 cells, and our previously described FVB/N-Tg(MMTV-PyVT)634Mul derived Met-1fvb2 and FVB/NTg(MMTV-PyVTY315F/Y322F) derived DB-7fvb2 cell lines. The NDLUCD cells have, so far, remained genetically and phenotypically stable over many generations, with consistent and reproducible results in immune intact preclinical cohorts.

Efficacy and safety of dihydroartemisinin-piperaquine for treatment of Plasmodium falciparum uncomplicated malaria in adult patients on antiretroviral therapy in Malawi and Mozambique: an open label non-randomized interventional trial.

BACKGROUND: HIV-infected individuals on antiretroviral therapy (ART) require treatment with artemisinin-based combination therapy (ACT) when infected with malaria. Dihydroartemisinin-piperaquine (DPQ) is recommended for treatment of Plasmodium falciparum malaria, but its efficacy and safety has not been evaluated in HIV-infected individuals on ART, among whom drug-drug interactions are expected. Day-42 adequate clinical and parasitological response (ACPR) and incidence of adverse events were assessed in HIV-infected individuals on non-nucleoside reverse transcriptase inhibitor-based ART (efavirenz and nevirapine) with uncomplicated P. falciparum malaria treated with dihydroartemisinin-piperaquine. METHODS: An open label single arm clinical trial was conducted in Malawi (Blantyre and Chikhwawa districts) and Mozambique (Manhiça district) involving patients aged 15-65 years with uncomplicated P. falciparum malaria who were on efavirenz-based or nevirapine-based ART. They received a directly-observed 3-day standard treatment of DPQ and were followed up until day 63 for malaria infection and adverse events. Day-42 PCR-corrected-ACPRs (95% confidence interval [CI]) were calculated for the intention-to-treat (ITT) population. RESULTS: The study enrolled 160 and 61 patients on efavirenz and nevirapine-based ART, with a baseline geometric mean (95% CI) parasite density of 2681 (1964-3661) and 9819 (6606-14,593) parasites/µL, respectively. The day-42 PCR-corrected ACPR (95% CI) was 99.4% (95.6-99.9%) in the efavirenz group and 100% in the nevirapine group. Serious adverse events occurred in 5.0% (8/160) and 3.3% (2/61) of the participants in the efavirenz and nevirapine group, respectively, but none were definitively attributable to DPQ. Cases of prolonged QT interval (> 60 ms from baseline) occurred in 31.2% (48/154) and 13.3% (8/60) of the patients on the efavirenz and nevirapine ART groups, respectively. These were not clinically significant and resolved spontaneously over time. As this study was not designed to compare the efficacy and safety of DPQ in the two ART groups, no formal statistical comparisons were made between the two ART groups. CONCLUSIONS: DPQ was highly efficacious and safe for the treatment of malaria in HIV-infected patients concurrently taking efavirenz- or nevirapine-based ART, despite known pharmacokinetic interactions between dihydroartemisinin-piperaquine and efavirenz- or nevirapine-based ART regimens. Trial registration Pan African Clinical Trials Registry (PACTR): PACTR201311000659400. Registered on 4 October 2013, https://pactr.samrc.ac.za/Search.aspx.

Efficacy and safety of artemether-lumefantrine as treatment for Plasmodium falciparum uncomplicated malaria in adult patients on efavirenz-based antiretroviral therapy in Zambia: an open label non-randomized interventional trial.

BACKGROUND: HIV-infected individuals on antiretroviral therapy (ART) require treatment with artemisinin-based combination therapy (ACT) when infected with malaria. Artemether-lumefantrine (AL) is the most commonly used ACT for treatment of falciparum malaria in Africa but there is limited evidence on the safety and efficacy of AL in HIV-infected individuals on ART, among whom drug-drug interactions are expected. Day-42 adequate clinical and parasitological response (ACPR) and incidence of adverse events was assessed in HIV-infected individuals on efavirenz-based ART with uncomplicated falciparum malaria treated with AL. METHODS: A prospective, open label, non-randomized, interventional clinical trial was conducted at St Paul's Hospital in northern Zambia, involving 152 patients aged 15-65 years with uncomplicated falciparum malaria, who were on efavirenz-based ART. They received a 3-day directly observed standard treatment of AL and were followed up until day 63. Day-42 polymerase chain reaction (PCR)-corrected ACPRs (95% confidence interval [CI]) were calculated for the intention-to-treat population. RESULTS: Enrolled patients had a baseline geometric mean (95% CI) parasite density of 1108 (841-1463) parasites/µL; 16.4% (25/152) of the participants had a recurrent malaria episode by day 42. However, PCR data was available for 17 out of the 25 patients who had malaria recurrence. Among all the 17 patients, PCR findings demonstrated malaria re-infection, making the PCR-adjusted day-42 ACPR 100% in the 144 patients who could be evaluated. Even when eight patients with missing PCR data were considered very conservatively as failures, the day-42 ACPR was over 94%. None of the participants, disease or treatment characteristics, including day-7 lumefantrine concentrations, predicted the risk of malaria recurrence by day 42. AL was well tolerated following administration. There were only two cases of grade 3 neutropaenia and one serious adverse event of lobar pneumonia, none of which was judged as probably related to intake of AL. CONCLUSIONS: AL was well tolerated and efficacious in treating uncomplicated falciparum malaria in HIV co-infected adults on efavirenz-based ART. However, a higher than anticipated proportion of participants experienced malaria re-infection, which highlights the need for additional malaria prevention measures in this sub-population after treatment with AL. Trial registration Pan African Clinical Trials Registry (PACTR): PACTR201311000659400. Registered on 4 October 2013. https://pactr.samrc.ac.za/Search.aspx.

Characterization and validation of an intra-fraction motion management system for masked-based radiosurgery.

PURPOSE: Characterize the intra-fraction motion management (IFMM) system found on the Gamma Knife Icon (GKI), including spatial accuracy, latency, temporal performance, and overall effect on delivered dose. METHODS: A phantom was constructed, consisting of a three-axis translation mount, a remote motorized flipper, and a thermoplastic sphere surrounding a radiation detector. An infrared marker was placed on the translation mount secured to the flipper. The spatial accuracy of the IFMM was measured via the translation mount in all Cartesian planes. The detector was centered at the radiation focal point. A remote signal was used to move the marker out of the IFMM tolerance and pause the beam. A two-channel electrometer was used to record the signals from the detector and the flipper when motion was signaled. These signals determined the latency and temporal performance of the GKI. RESULTS: The spatial accuracy of the IFMM was found to be <0.1 mm. The measured latency was <200 ms. The dose difference with five interruptions was <0.5%. CONCLUSION: This work provides a quantitative characterization of the GKI IFMM system as required by the Nuclear Regulatory Commission. This provides a methodology for GKI users to satisfy these requirements using common laboratory equipment in lieu of a commercial solution.

Obatoclax, a BH3 Mimetic, Enhances Cisplatin-Induced Apoptosis and Decreases the Clonogenicity of Muscle Invasive Bladder Cancer Cells via Mechanisms That Involve the Inhibition of Pro-Survival Molecules as Well as Cell Cycle Regulators.

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.