A Safe Framework for Quantitative In Vivo Human Evaluation of Image Guidance.

Goal: We present a new framework for in vivo image guidance evaluation and provide a case study on robotic partial nephrectomy. Methods: This framework (called the "bystander protocol") involves two surgeons, one who solely performs the therapeutic process without image guidance, and another who solely periodically collects data to evaluate image guidance. This isolates the evaluation from the therapy, so that in-development image guidance systems can be tested without risk of negatively impacting the standard of care. We provide a case study applying this protocol in clinical cases during robotic partial nephrectomy surgery. Results: The bystander protocol was performed successfully in 6 patient cases. We find average lesion centroid localization error with our IGS system to be 6.5 mm in vivo compared to our prior result of 3.0 mm in phantoms. Conclusions: The bystander protocol is a safe, effective method for testing in-development image guidance systems in human subjects.

CAR+ and CAR- T cells share a differentiation trajectory into an NK-like subset after CD19 CAR T cell infusion in patients with B cell malignancies.

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients' lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR- T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR+ T cells and CAR- T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR+ and CAR- T cells. These results suggest that non-CAR-derived signals can provide information about patients' immune recovery and be used as correlate of clinically relevant parameters.

HIVepsilon-seq-scalable characterization of intact persistent proviral HIV reservoirs in women.

IMPORTANCE: The lack of a reliable method to accurately detect when replication-competent HIV has been cleared is a major challenge in developing a cure. This study introduces a new approach called the HIVepsilon-seq (HIVε-seq) assay, which uses long-read sequencing technology and bioinformatics to scrutinize the HIV genome at the nucleotide level, distinguishing between defective and intact HIV. This study included 30 participants on antiretroviral therapy, including 17 women, and was able to discriminate between defective and genetically intact viruses at the single DNA strand level. The HIVε-seq assay is an improvement over previous methods, as it requires minimal sample, less specialized lab equipment, and offers a shorter turnaround time. The HIVε-seq assay offers a promising new tool for researchers to measure the intact HIV reservoir, advancing efforts towards finding a cure for this devastating disease.

Patient-specific, touch-based registration during robotic, image-guided partial nephrectomy.

Image-guidance during partial nephrectomy enables navigation within the operative field alongside a 3-dimensional roadmap of renal anatomy generated from patient-specific imaging. Once a process is performed by the human mind, the technology will allow standardization of the task for the benefit of all patients undergoing robot-assisted partial nephrectomy. Any surgeon will be able to visualize the kidney and key subsurface landmarks in real-time within a 3-dimensional simulation, with the goals of improving operative efficiency, decreasing surgical complications, and improving oncologic outcomes. For similar purposes, image-guidance has already been adopted as a standard of care in other surgical fields; we are now at the brink of this in urology. This review summarizes touch-based approaches to image-guidance during partial nephrectomy, as the technology begins to enter in vivo human evaluation. The processes of segmentation, localization, registration, and re-registration are all described with seamless integration into the da Vinci surgical system; this will facilitate clinical adoption sooner.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Accuracy of Touch-Based Registration During Robotic Image-Guided Partial Nephrectomy Before and After Tumor Resection in Validated Phantoms.

Aim: Image-guided surgery (IGS) allows for accurate, real-time localization of subsurface critical structures during surgery. No prior IGS systems have described a feasible method of intraoperative reregistration after manipulation of the kidney during robotic partial nephrectomy (PN). We present a method for seamless reregistration during IGS and evaluate accuracy before and after tumor resection in two validated kidney phantoms. Materials and Methods: We performed robotic PN on two validated kidney phantoms-one with an endophytic tumor and one with an exophytic tumor-with our IGS system utilizing the da Vinci Xi robot. Intraoperatively, the kidney phantoms' surfaces were digitized with the da Vinci robotic manipulator via a touch-based method and registered to a three-dimensional segmented model created from cross-sectional CT imaging of the phantoms. Fiducial points were marked with a surgical marking pen and identified after the initial registration using the robotic manipulator. Segmented images were displayed via picture-in-picture in the surgeon console as tumor resection was performed. After resection, reregistration was performed by reidentifying the fiducial points. The accuracy of the initial registration and reregistration was compared. Results: The root mean square (RMS) averages of target registration error (TRE) were 2.53 and 4.88 mm for the endophytic and exophytic phantoms, respectively. IGS enabled resection along preplanned contours. Specifically, the RMS averages of the normal TRE over the entire resection surface were 0.75 and 2.15 mm for the endophytic and exophytic phantoms, respectively. Both tumors were resected with grossly negative margins. Point-based reregistration enabled instantaneous reregistration with minimal impact on RMS TRE compared with the initial registration (from 1.34 to 1.70 mm preresection and from 1.60 to 2.10 mm postresection). Conclusions: We present a novel and accurate registration and reregistration framework for use during IGS for PN with the da Vinci Xi surgical system. The technology is easily integrated into the surgical workflow and does not require additional hardware.

Comparing the accuracy of the da Vinci Xi and da Vinci Si for image guidance and automation.

BACKGROUND: Current laparoscopic surgical robots are teleoperated, which requires high fidelity differential motions but does not require absolute accuracy. Emerging applications, including image guidance and automation, require absolute accuracy. The absolute accuracy of the da Vinci Xi robot has not yet been characterized or compared to the Si system, which is now being phased out. This study compares the accuracy of the two. METHODS: We measure robot tip positions and encoder values assessing accuracy with and without robot calibration. RESULTS: The Si is accurate if the setup joints are not moved but loses accuracy otherwise. The Xi is always accurate. CONCLUSION: The Xi can achieve submillimetric average error. Calibration improves accuracy, but excellent baseline accuracy of the Xi means that calibration may not be needed for some applications. Importantly, the external tracking systems needed to account for setup joint error in the Si are no longer required with the Xi.

Toward Practical and Accurate Touch-Based Image Guidance for Robotic Partial Nephrectomy.

Partial nephrectomy involves removing a tumor while sparing surrounding healthy kidney tissue. Compared to total kidney removal, partial nephrectomy improves outcomes for patients but is underutilized because it is challenging to accomplish minimally invasively, requiring accurate spatial awareness of unseen subsurface anatomy. Image guidance can enhance spatial awareness by displaying a 3D model of anatomical relationships derived from medical imaging information. It has been qualitatively suggested that the da Vinci robot is well suited to facilitate image guidance through touch-based registration. In this paper we validate and advance this concept toward real-world use in several important ways. First, we contribute the first quantitative accuracy evaluation of touch-based registration with the da Vinci. Next, we demonstrate real-time touch-based registration and display of medical images for the first time. Lastly, we perform the first experiments validating use of touch-based image guidance to improve a surgeon's ability to localize subsurface anatomical features in a geometrically realistic phantom.

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.

Pharmacokinetics of fluoxetine in elderly men and women.

Fluoxetine is widely prescribed as an antidepressant for geriatric patients. Despite a large scientific literature describing its efficacy and safety, there are few published data describing the pharmacokinetics of fluoxetine in the elderly. Given the common practice of polypharmacy in this population, additional pharmacokinetic information in elderly men and women is needed so that physicians can better assess potential drug-drug interactions. Twenty-five subjects, men and women between ages 65 and 83, received 20 mg of fluoxetine for 1 week followed by 40 mg for 5 weeks. Serum fluoxetine levels were measured during the period of drug administration and for 8 weeks after. The plasma concentration of fluoxetine and norfluoxetine in our subjects was higher than previously reported in the literature. Elderly women had a significantly higher serum level of norfluoxetine than men. The terminal half-life of norfluoxetine was longer in patients over age 75; elderly women had a significantly slower rate of norfluoxetine elimination than similarly aged men.