Label-aware distance mitigates temporal and spatial variability for clustering and visualization of single-cell gene expression data.

Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. The batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Label-Aware Distance (LAD), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate LAD on simulated data as well as apply it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). LAD provides better cell embedding than state-of-the-art batch correction methods on longitudinal datasets. It can be used in distance-based clustering and visualization methods to combine the power of multiple samples to help make biological findings.

Transcriptional and phenotypic heterogeneity underpinning venetoclax resistance in AML.

The venetoclax BCL2 inhibitor in combination with hypomethylating agents represents a cornerstone of induction therapy for older AML patients, unfit for intensive chemotherapy. Like other targeted therapies, venetoclax-based therapies suffer from innate and acquired resistance. While several mechanisms of resistance have been identified, the heterogeneity of resistance mechanism across patient populations is poorly understood. Here we utilized integrative analysis of transcriptomic and ex-vivo drug response data in AML patients to identify four transcriptionally distinct VEN resistant clusters (VR_C1-4), with distinct phenotypic, genetic and drug response patterns. VR_C1 was characterized by enrichment for differentiated monocytic- and cDC-like blasts, transcriptional activation of PI3K-AKT-mTOR signaling axis, and energy metabolism pathways. They showed sensitivity to mTOR and CDK inhibition. VR_C2 was enriched for NRAS mutations and associated with distinctive transcriptional suppression of HOX expression. VR_C3 was characterized by enrichment for TP53 mutations and higher infiltration by cytotoxic T cells. This cluster showed transcriptional expression of erythroid markers, suggesting tumor cells mimicking erythroid differentiation, activation of JAK-STAT signaling, and sensitivity to JAK inhibition, which in a subset of cases synergized with venetoclax. VR_C4 shared transcriptional similarities with venetoclax-sensitive patients, with modest over-expression of interferon signaling. They were also characterized by high rates of DNMT3A mutations. Finally, we projected venetoclax-resistance states onto single cells profiled from a patient who relapsed under venetoclax therapy capturing multiple resistance states in the tumor and shifts in their abundance under venetoclax selection, suggesting that single tumors may consist of cells mimicking multiple VR_Cs contributing to intra-tumor heterogeneity. Taken together, our results provide a strategy to evaluate inter- and intra-tumor heterogeneity of venetoclax resistance mechanisms and provide insights into approaches to navigate further management of patients who failed therapy with BCL2 inhibitors.

Batch-Corrected Distance Mitigates Temporal and Spatial Variability for Clustering and Visualization of Single-Cell Gene Expression Data.

Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. The batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Batch-Corrected Distance (BCD), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate BCD on simulated data as well as applied it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). BCD achieves more accurate clusters and better visualizations than state-of-the-art batch correction methods on longitudinal datasets. BCD can be directly integrated with most clustering and visualization methods to enable more scientific findings.

Selective parenchymal clamping in open partial nephrectomy: Early experience with a low cost, reusable novel parenchymal clamp.

Objectives: The objective of this study is to present our initial experience with a novel parenchymal clamp (NPC) developed to allow partial nephrectomies (PN) to be performed without whole kidney ischaemia. We compare patients who underwent PN with the NPC with those undergoing standard PNs. Methods: The NPC applies pressure only to the portion of the parenchyma containing the small renal mass (≤3.5 cm) and interrupts regional blood flow.A retrospective analysis was conducted on patients that underwent open PN within our unit. Minimum follow-up was 12 months. Patient and disease characteristics, perioperative outcomes and renal function estimated Glomerular Filtration Rate (eGFR) were compared. Results: Data were collected on 63 patients, of whom 33 had their procedure performed with the NPC. Demographic characteristics and comorbidity profiles were not significantly different between groups (p between 1.0 and 0.08). RENAL nephrometry scores were 5.6 in the NPC group versus 6.2 in the standard PN group (p = 0.146).Perioperative, operative and postoperative data did not show significant differences. There was no difference in the rates of Clavien-Dindo III or above complications between the two groups (NPC 3/33 vs. standard PN 5/30, p = 0.416). There was also no statistically significant difference to changes in renal functional at 12 months (change -5.2 and -6.4, p = 0.712). Conclusions: Despite the limited sample size and follow-up, this study establishes the safety of the NPC. In the future, we intend to perform a prospective study on the laparoscopic version.

Metastatic renal cell carcinoma presenting as jaundice with biliary and gastric outlet obstruction. A case report.

Renal cell carcinoma (RCC) can be an aggressive malignancy that has a propensity to spread to atypical locations, most commonly to lung, bone, lymph node. RCC presenting as obstructive jaundice with gastric outlet obstruction has rarely been cited in literature. This study presents a case of advanced RCC in a patient with obstructive jaundice and associated gastric outlet obstruction from a large right renal RCC with malignant retrocaval lymphadenopathy invading the duodenum and distal common bile duct. The patient underwent anterograde stenting of the biliary system via a percutaneous transhepatic cholangiography and an insertion of a duodenal stent. Immunotherapy was commenced and the patient was discharged home. This case highlights the importance of a multi-disciplinary team approach to the management of a complex surgical patient.

Ab initio spillover compensation in mass cytometry data.

Signal intensity measured in a mass cytometry (CyTOF) channel can often be affected by the neighboring channels due to technological limitations. Such signal artifacts are known as spillover effects and can substantially limit the accuracy of cell population clustering. Current approaches reduce these effects by using additional beads for normalization purposes known as single-stained controls. While effective in compensating for spillover effects, incorporating single-stained controls can be costly and require customized panel design. This is especially evident when executing large-scale immune profiling studies. We present a novel statistical method, named CytoSpill that independently quantifies and compensates the spillover effects in CyTOF data without requiring the use of single-stained controls. Our method utilizes knowledge-guided modeling and statistical techniques, such as finite mixture modeling and sequential quadratic programming, to achieve optimal error correction. We evaluated our method using five publicly available CyTOF datasets obtained from human peripheral blood mononuclear cells (PBMCs), C57BL/6J mouse bone marrow, healthy human bone marrow, chronic lymphocytic leukemia patient, and healthy human cord blood samples. In the PBMCs with known ground truth, our method achieved comparable results to experiments that incorporated single-stained controls. In datasets without ground-truth, our method not only reduced spillover on likely affected markers, but also led to the discovery of potentially novel subpopulations expressing functionally meaningful, cluster-specific markers. CytoSpill (developed in R) will greatly enhance the execution of large-scale cellular profiling of tumor immune microenvironment, development of novel immunotherapy, and the discovery of immune-specific biomarkers. The implementation of our method can be found at https://github.com/KChen-lab/CytoSpill.git.

Batch-Corrected Distance Mitigates Temporal and Spatial Variability for Clustering and Visualization of Single-Cell Gene Expression Data.

Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. Batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Batch-Corrected Distance (BCD), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate BCD on a simulated data as well as applied it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). BCD achieves more accurate clusters and better visualizations than state-of-the-art batch correction methods on longitudinal datasets. BCD can be directly integrated with most clustering and visualization methods to enable more scientific findings.

Integrated transcriptomic-genomic tool Texomer profiles cancer tissues.

Profiling of both the genome and the transcriptome promises a comprehensive, functional readout of a tissue sample, yet analytical approaches are required to translate the increased data dimensionality, heterogeneity and complexity into patient benefits. We developed a statistical approach called Texomer ( https://github.com/KChen-lab/Texomer ) that performs allele-specific, tumor-deconvoluted transcriptome-exome integration of autologous bulk whole-exome and transcriptome sequencing data. Texomer results in substantially improved accuracy in sample categorization and functional variant prioritization.

FBXW7 regulates a mitochondrial transcription program by modulating MITF.

FBXW7 is well characterized as a tumor suppressor in many human cancers including melanoma; however, the mechanisms of tumor-suppressive function have not been fully elucidated. We leveraged two distinct RNA sequencing datasets: human melanoma cell lines (n = 10) with control versus silenced FBXW7 and a cohort of human melanoma tumor samples (n = 51) to define the transcriptomic fingerprint regulated by FBXW7. Here, we report that loss of FBXW7 enhances a mitochondrial gene transcriptional program that is dependent on MITF in human melanoma and confers poor patient outcomes. MITF is a lineage-specific master regulator of melanocytes and together with PGC-1alpha is a marker for melanoma subtypes with dependence for mitochondrial oxidative metabolism. We found that inactivation of FBXW7 elevates MITF protein levels in melanoma cells. In vitro studies examining loss of FBXW7 and MITF alone or in combination showed that FBXW7 is an upstream regulator for the MITF/PGC-1 signaling.

Transcriptional dissection of melanoma identifies a high-risk subtype underlying TP53 family genes and epigenome deregulation.

BACKGROUND: Melanoma is a heterogeneous malignancy. We set out to identify the molecular underpinnings of high-risk melanomas, those that are likely to progress rapidly, metastasize, and result in poor outcomes. METHODS: We examined transcriptome changes from benign states to early-, intermediate-, and late-stage tumors using a set of 78 treatment-naive melanocytic tumors consisting of primary melanomas of the skin and benign melanocytic lesions. We utilized a next-generation sequencing platform that enabled a comprehensive analysis of protein-coding and -noncoding RNA transcripts. RESULTS: Gene expression changes unequivocally discriminated between benign and malignant states, and a dual epigenetic and immune signature emerged defining this transition. To our knowledge, we discovered previously unrecognized melanoma subtypes. A high-risk primary melanoma subset was distinguished by a 122-epigenetic gene signature ("epigenetic" cluster) and TP53 family gene deregulation (TP53, TP63, and TP73). This subtype associated with poor overall survival and showed enrichment of cell cycle genes. Noncoding repetitive element transcripts (LINEs, SINEs, and ERVs) that can result in immunostimulatory signals recapitulating a state of "viral mimicry" were significantly repressed. The high-risk subtype and its poor predictive characteristics were validated in several independent cohorts. Additionally, primary melanomas distinguished by specific immune signatures ("immune" clusters) were identified. CONCLUSION: The TP53 family of genes and genes regulating the epigenetic machinery demonstrate strong prognostic and biological relevance during progression of early disease. Gene expression profiling of protein-coding and -noncoding RNA transcripts may be a better predictor for disease course in melanoma. This study outlines the transcriptional interplay of the cancer cell's epigenome with the immune milieu with potential for future therapeutic targeting. FUNDING: National Institutes of Health (CA154683, CA158557, CA177940, CA087497-13), Tisch Cancer Institute, Melanoma Research Foundation, the Dow Family Charitable Foundation, and the Icahn School of Medicine at Mount Sinai.

Case-control study of factors that trigger inflammatory bowel disease flares.

AIM: To explore the association between inflammatory bowel diseases (IBD) flares and potential triggers. METHODS: Patients evaluated for an acute flare of IBD by a gastroenterologist at the Dallas VA Medical Center were invited to participate, as were a control group of patients with IBD in remission. Patients were systematically queried about nonsteroidal anti-inflammatory drug use, antibiotic use, stressful life events, cigarette smoking, medication adherence, infections, and travel in the preceding 3 mo. Disease activity scores were calculated for each patient at the time of enrollment and each patient's chart was reviewed. Multivariate regression analysis was performed. RESULTS: A total of 134 patients with IBD (63 with Crohn's disease, 70 with ulcerative colitis, and 1 with indeterminate colitis) were enrolled; 66 patients had flares of their IBD and 68 were controls with IBD in remission (for Crohn's patients, average Crohn's disease activity index was 350 for flares vs 69 in the controls; for UC patients, Mayo score was 7.6 for flares vs 1 for controls in those with full Mayo available and 5.4p for flares vs 0.1p for controls in those with partial Mayo score). Only medication non-adherence was significantly more frequent in the flare group than in the control group (48.5% vs 29.4%, P = 0.03) and remained significant on multivariate analysis (OR = 2.86, 95%CI: 1.33-6.18). On multivariate regression analysis, immunomodulator use was found to be associated with significantly lower rates of flare (OR = 0.40, 95%CI: 0.19-0.86). CONCLUSION: In a study of potential triggers for IBD flares, medication non-adherence was significantly associated with flares. These findings are incentive to improve medication adherence.

The prophylactic placement of hemoclips to prevent delayed post-polypectomy bleeding: an unnecessary practice? A case control study.

BACKGROUND: With the recent, widespread availability of endoscopic hemoclips, it has become common clinical practice to apply hemoclips to some non-bleeding polypectomy sites "prophylactically" to prevent delayed post-polypectomy bleeding (PPB). Few published data support this practice, however. AIM: The aim of this study was to compare rates of delayed PPB in matched patients who had polypectomies performed with and without the prophylactic placement of hemoclips. METHODS: We reviewed medical records of patients who had elective colonoscopy at our VA Medical Center between July 2008 and December 2009. We identified patients who had hemoclips applied prophylactically (cases) and compared their rate of delayed PPB within 30 days to that of patients who had polypectomy without hemoclipping (controls). Controls were matched 1:1 to cases based on age and on factors known to contribute to the risk of PPB including polyp size, morphology, technique of polyp removal, number of polyps removed, and use of anticoagulants. RESULTS: We identified 184 patients (cases) who underwent prophylactic hemoclipping and 184 well-matched controls. An average of 3.8 polyps per patient were removed in the case group compared to 3.3 polyps per patient in controls (p = 0.6). Delayed PPB occurred in three patients in the prophylactic hemoclip group and in one patient in the control group (1.6 vs. 0.5 %, p = 0.62). CONCLUSIONS: We found no significant difference in the rate of delayed PPB between patients who had prophylactic hemoclipping of polypectomy sites and a well-matched control group of patients who had polypectomy without prophylactic hemoclipping. These data call into question the expensive practice of prophylactic hemoclipping.

Helicobacter gastritis induces changes in the oxyntic mucosa indistinguishable from the effects of proton pump inhibitors.

A causal relationship between oxyntic glands dilatation with protruding parietal cells, referred to as proton pump inhibitor (PPI) effects, and PPI use has been suspected but not established. We designed this study to evaluate the association between these changes and the use of PPIs and histamine2-receptor blockers (H2-blockers). We obtained five Sydney System-compliant biopsy specimens from patients recruited into a therapeutic trial for H. pylori. Medication history with details on PPI and H2-blockers use was collected. Two blinded pathologists graded gastritis and the intensity of putative PPI effects using a 0 to 3 scale. PPI and H2-blocker use was then disclosed and the accuracy of pathologists' assessment was analyzed. There were 138 H. pylori-negative and 104 positive patients. In H. pylori-negative patients the histologic assessment for PPI use had 77.5% sensitivity and 51.8% specificity, with a positive predictive value of 86.9% and a negative predictive value of 35.9%. In H. pylori-positive patients, sensitivity was 74.1% and specificity 26.1%. Positive and negative predictive values were 55.8% and 44.4%, respectively. Neither glandular dilatations nor parietal cell protrusions related to H2-blocker use. We conclude that these changes are associated with PPI use only in H. pylori-negative subjects. In H. pylori gastritis, so-called PPI-effects were equally prevalent in PPI-users and non-users, indicating that other factors are involved in the induction of oxyntic cell hyperplasia. We suggest that comments regarding the supposed evidence of PPI use are too often wrong to be useful and should be avoided in the diagnosis of gastric biopsy specimens.

Clinical implications of histologic abnormalities in colonic biopsy specimens from patients with ulcerative colitis in clinical remission.

BACKGROUND: For patients with ulcerative colitis (UC) who have colonoscopy while their disease is in clinical remission, the clinical implications of finding histologic abnormalities of colitis are not clear. METHODS: We reviewed the medical records of patients with UC who had elective colonoscopy at our VA Medical Center while their UC was in clinical remission and who had at least 6 months of follow-up data available. The Mayo endoscopic subscore was used to assess endoscopic disease activity. Biopsies were evaluated for specific changes in the following general categories: acute inflammation, chronic inflammation, epithelial damage, and architectural distortion. RESULTS: We identified 51 patients with UC who had a total of 84 colonoscopies (at least 1 year apart) while they were in clinical remission. Forty colonoscopies revealed no endoscopic activity (Mayo subscore 0) and 44 showed endoscopic activity (Mayo subscore 1, 2, or 3). Although flares were approximately twice as common in patients with endoscopic activity as in those with an endoscopically normal mucosa, the difference was not statistically significant. On histologic evaluation, in contrast, the presence of basal lymphoplasmacytosis, basally located lymphoid aggregates, erosions and/or ulcerations of the epithelium, or moderate to marked architectural distortion all were significant predictors of clinical flares by 6 and 12 months. CONCLUSIONS: For patients with UC who have colonoscopy while their disease is in clinical remission, colonic biopsy seems to provide important prognostic information beyond that provided by the endoscopic assessment of disease activity alone.

Low rate of postpolypectomy bleeding among patients who continue thienopyridine therapy during colonoscopy.

BACKGROUND & AIMS: It is not clear whether the cardiovascular risk of discontinuing treatment with antiplatelet agents, specifically the thienopyridines, before elective colonoscopy outweighs the risks of postpolypectomy bleeding (PPB). We studied the rate of PPB in patients who continue thienopyridine therapy during colonoscopy. METHODS: We performed a prospective study of 516 patients not taking warfarin who received polypectomies during elective colonoscopies; 219 were receiving thienopyridines, and 297 were not (controls). The occurrence of immediate PPB and delayed PPB was recorded. Delayed PPB was categorized as clinically important if it resulted in repeat colonoscopy, hospitalization, or blood transfusion. RESULTS: Patients receiving thienopyridines were older and had significantly more comorbid diseases than controls; the mean number of polyps removed per patient was significantly higher (3.9 vs 2.9) in the thienopyridine group. Immediate PPB developed in 16 patients in the thienopyridine group (7.3%) and in 14 in the control group (4.7%, P = .25). Among patients who completed a 30-day follow-up analysis (96% of patients enrolled), clinically important, delayed bleeding occurred in 2.4% of patients receiving thienopyridines and in none of the controls (P = .01). All PPB events in both groups were resolved without surgery, angiography, or death. CONCLUSIONS: Although a significantly higher percentage of patients who continue thienopyridine therapy during colonoscopy and polypectomy develop clinically important delayed PPB than patients who discontinue therapy, the rate of PPB events is low (2.4%), and all are resolved without sequelae. The risk for catastrophic cardiovascular risks among patients who discontinue thienopyridine therapy before elective colonoscopies could therefore exceed the risks of PPB. ClinicalTrials.gov, Number NCT01647568.

Prone positioning of obese patients for colonoscopy results in shortened cecal intubation times: a randomized trial.

BACKGROUND: Obesity is a risk factor for colorectal cancer, and colonoscopy can be technically challenging in obese patients. It has been proposed (with little supporting data) that prone positioning of obese patients might facilitate a difficult colonoscopy. AIM: The aim of this study was to determine if starting colonoscopy in the prone position for obese patients decreases cecal intubation times. METHODS: This was a prospective, randomized study conducted at the North Texas VA Medical Center. Patients with a body mass index of ≥30 kg/m(2) undergoing elective colonoscopy were randomized 1:1 to either initial prone positioning or standard, left-lateral positioning. The outcome measurements were cecal intubation time, frequency of repositioning, sedative medications used, reports of pain, complications, and procedure tolerability. RESULTS: Fifty patients were randomized to have colonoscopy starting in the standard, left-lateral decubitus position, and 51 to the prone position. The average cecal intubation time for the standard group was 550 vs. 424 s in the prone group (p = 0.03). Patient repositioning was used in 28 % of patients in the standard group versus 8 % in the prone group (p = 0.009). There was no difference in subjective reports of pain between groups (p = 0.95) or in average pain scores (p = 0.79). Follow-up interviews were conducted in 93 % of patients, all of whom said that they would be willing to have repeat colonoscopy in the same position. CONCLUSIONS: Performance of colonoscopy in the prone position for obese patients results in significantly shorter cecal intubation times and decreased need for patient repositioning. Prone positioning is well accepted and does not significantly increase procedure-related discomfort.