Highly Multiplexed Spatially Resolved Proteomic and Transcriptional Profiling of the Glioblastoma Microenvironment Using Archived Formalin-Fixed Paraffin-Embedded Specimens.

Glioblastoma is a heterogeneous tumor for which effective treatment options are limited and often insufficient. Few studies have examined the intratumoral transcriptional and proteomic heterogeneity of the glioblastoma microenvironment to characterize the spatial distribution of potential molecular and cellular therapeutic immunooncology targets. We applied an integrated multimodal approach comprised of NanoString GeoMx Digital Spatial Profiling, single-cell RNA-seq (scRNA-seq), and expert neuropathologic assessment to characterize archival formalin-fixed paraffin-embedded glioblastoma specimens. Clustering analysis and spatial cluster maps highlighted the intratumoral heterogeneity of each specimen. Mixed cell deconvolution analysis revealed that neoplastic and vascular cells were the prominent cell types throughout each specimen, with macrophages, oligodendrocyte precursors, neurons, astrocytes, and oligodendrocytes present in lower abundance and illustrated the regional distribution of the respective cellular enrichment scores. The spatial resolution of the actionable immunotherapeutic landscape showed that robust B7H3 gene and protein expression was broadly distributed throughout each specimen and identified STING and VISTA as potential targets. Lastly, we uncovered remarkable variability in VEGFA expression and discovered unanticipated associations between VEGFA, endothelial cell markers, hypoxia, and the expression of immunoregulatory genes, indicative of regionally distinct immunosuppressive microdomains. This work provides an early demonstration of the ability of an integrated panel-based spatial biology approach to characterize and quantify the intrinsic molecular heterogeneity of the glioblastoma microenvironment.

Longitudinal transcriptional analysis of peripheral blood leukocytes in COVID-19 convalescent donors.

BACKGROUND: SARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19). METHODS: Using the nCounter platform, we compared transcriptomic profiles of 162 COVID-19 convalescent donors (CCD) and 40 healthy donors (HD). 69 of the 162 CCDs had two or more time points sampled. RESULTS: After eliminating the effects of demographic factors, we found extensive differential gene expression up to 241 days into the convalescent period. The differentially expressed genes were involved in several pathways, including virus-host interaction, interleukin and JAK-STAT signaling, T-cell co-stimulation, and immune exhaustion. A subset of 21 CCD samples was found to be highly "perturbed," characterized by overexpression of PLAU, IL1B, NFKB1, PLEK, LCP2, IRF3, MTOR, IL18BP, RACK1, TGFB1, and others. In addition, one of the clusters, P1 (n = 8) CCD samples, showed enhanced TCR diversity in 7 VJ pairs (TRAV9.1_TCRVA_014.1, TRBV6.8_TCRVB_016.1, TRAV7_TCRVA_008.1, TRGV9_ENST00000444775.1, TRAV18_TCRVA_026.1, TRGV4_ENST00000390345.1, TRAV11_TCRVA_017.1). Multiplexed cytokine analysis revealed anomalies in SCF, SCGF-b, and MCP-1 expression in this subset. CONCLUSIONS: Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed following recovery from COVID-19 infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT04360278 Registered April 24, 2020.

Accuracy evaluations of three ruptured abdominal aortic aneurysm mortality risk scores using an independent dataset.

OBJECTIVE: To date, no single scoring system for predicting 30-day mortality in patients with ruptured abdominal aortic aneurysms (rAAAs) has been endorsed by any vascular society or proven to definitively predict treatment futility. Three recently developed scoring systems for predicting 30-day mortality in patients with rAAA have been validated by their respective institutional data. The purpose of this study was to evaluate the accuracy of these rAAA mortality risk scores using an independent community hospital dataset. METHODS: Consecutive patients presenting with rAAA at Saint Joseph Hospital between January 1, 2009, and February 28, 2017, were used for validation. Logistic regression analysis was used to evaluate the association between risk score and odds of death. Confidence intervals were calculated using the Wilson method. Comparisons were made between models by calculating the area under the receiver operating characteristic (AUC) curves. RESULTS: Complete data from 38 patients was used for accuracy evaluation. The AUCs for the Dutch Aneurysm Score, Harborview Medical Center score, and Vascular Surgery Group of New England (VSGNE) score were 0.762, 0.792, and 0.860, respectively, for all patients. When evaluating 30-day mortality for patients undergoing ruptured endovascular aneurysm repair, the scores were 0.802, 0.893, and 0.927, respectively. The difference between scores did not reach statistical significance. All three indexes significantly associated with the mortality rate using logistic regression. CONCLUSIONS: Each risk score accurately predicted 30-day mortality using the independent dataset. The results suggest that the VSGNE score is the most accurate; however, differences in accuracy between each scoring system did not reach statistical significance. The Harborview Medical Center scoring system is based only on preoperative variables. Although the VSGNE score had the highest AUC in this analysis, it is dependent on intraoperative variables. The authors favor a single risk assessment tool, with consensus vascular societal approval, that incorporates preoperative variables and includes a tool for the prediction of treatment futility.

Preoperative risk score for the prediction of mortality after repair of ruptured abdominal aortic aneurysms.

OBJECTIVE: Even in the ruptured endovascular aneurysm repair first era, there are still patients who will not survive their ruptured abdominal aortic aneurysm (rAAA). All previously published mortality risk scores include intraoperative variables and are not helpful with the decision to operate or in providing preoperative patient and family counseling. The purpose of this study was to develop a practical preoperative risk score to predict mortality after repair of rAAA. METHODS: Data of all patients with rAAA presenting between January 1, 2002, and October 31, 2013, were collected. Logistic regression was used to evaluate predictive variables both univariately and jointly, and the results of multivariate models guided the definition of the final simplified scoring algorithm. RESULTS: There were 303 patients who presented during the study period. Sixteen patients died in the emergency department, en route to surgery, or after choosing comfort care. Preoperative variables most predictive of mortality were age >76 years (odds ratio [OR], 2.11; confidence interval [CI], 1.47-4.97; P = .011), creatinine concentration >2.0 mg/dL (OR, 3.66; CI, 1.85-7.24; P < .001), pH <7.2 (OR, 2.58; CI, 1.27-5.24; P = .009), and systolic blood pressure ever <70 mm Hg (OR, 2.70; CI, 1.46-4.97; P = .002). Assigning 1 point for each variable, patients were stratified according to the preoperative rAAA mortality risk score (range, 0-4). For all repairs, at 30 days, patients with 1 point suffered 22% mortality; 2 points, 69% mortality; and 3 points, 80% mortality. All patients with 4 points died. There was a mortality benefit for ruptured endovascular aneurysm repair across all categories. CONCLUSIONS: Our rAAA mortality risk score is based on four variables readily assessed in the emergency department and allows accurate prediction of 30-day mortality after repair of rAAAs. It also has a direct impact on clinical decision-making by adding prognostic information to the decision to transfer patients to tertiary care centers and aiding in preoperative discussions with patients and their families.

Reliability, Validity, and Responsiveness of InFLUenza Patient-Reported Outcome (FLU-PRO©) Scores in Influenza-Positive Patients.

OBJECTIVES: To assess the reliability, validity, and responsiveness of InFLUenza Patient-Reported Outcome (FLU-PRO©) scores for quantifying the presence and severity of influenza symptoms. METHODS: An observational prospective cohort study of adults (≥18 years) with influenza-like illness in the United States, the United Kingdom, Mexico, and South America was conducted. Participants completed the 37-item draft FLU-PRO daily for up to 14 days. Item-level and factor analyses were used to remove items and determine factor structure. Reliability of the final tool was estimated using Cronbach α and intraclass correlation coefficients (2-day reliability). Convergent and known-groups validity and responsiveness were assessed using global assessments of influenza severity and return to usual health. RESULTS: Of the 536 patients enrolled, 221 influenza-positive subjects comprised the analytical sample. The mean age of the patients was 40.7 years, 60.2% were women, and 59.7% were white. The final 32-item measure has six factors/domains (nose, throat, eyes, chest/respiratory, gastrointestinal, and body/systemic), with a higher order factor representing symptom severity overall (comparative fit index = 0.92; root mean square error of approximation = 0.06). Cronbach α was high (total = 0.92; domain range = 0.71-0.87); test-retest reliability (intraclass correlation coefficient, day 1-day 2) was 0.83 for total scores and 0.57 to 0.79 for domains. Day 1 FLU-PRO domain and total scores were moderately to highly correlated (≥0.30) with Patient Global Rating of Flu Severity (except nose and throat). Consistent with known-groups validity, scores differentiated severity groups on the basis of global rating (total: F = 57.2, P < 0.001; domains: F = 8.9-67.5, P < 0.001). Subjects reporting return to usual health showed significantly greater (P < 0.05) FLU-PRO score improvement by day 7 than did those who did not, suggesting score responsiveness. CONCLUSIONS: Results suggest that FLU-PRO scores are reliable, valid, and responsive to change in influenza-positive adults.

The Incidence of Ischemic Colitis after Repair of Ruptured Abdominal Aortic Aneurysms Is Decreasing in the Endovascular Era.

BACKGROUND: Ischemic colitis (IC) is a well-described complication of ruptured abdominal aortic aneurysms (rAAAs). The purpose of this study was to compare the incidence of IC in patients with rAAA undergoing open repair (OR) versus endovascular aneurysm repair (EVAR) at a single institution. In addition, we analyzed the incidence of IC before and after the implementation of a formal rupture AAA protocol (rEVAR protocol). METHODS: A retrospective analysis of prospectively collected data on all patients presenting with rAAA to our institution between January 2002 and October 2013 was performed. Variables were analyzed for association with IC. Comparisons were made using Pearson's chi-squared test or Fisher's exact test for categorical variables, Student's t-test for continuous variables, and logistic regression for multivariate analysis. Significance was set at P < 0.05. RESULTS: Three hundred three patients with rAAA presented over the 10-year study period. One hundred ninety-one patients underwent OR and 89 patients underwent endovascular repair. Twenty-three patients died either in the emergency department, en route to the operating room, or after choosing comfort care. Predictive factors of IC included estimated blood loss, corresponding need for resuscitation, and duration of procedure. Of patients who underwent OR, the rate of IC was 21% (40/191). This was significantly higher than patients who underwent EVAR, 7% (6/89), P < 0.05. The type of intervention did not influence 30-day mortality in patients with IC. However, only 17% (1/6) of patients who had IC following EVAR required colectomy versus 48% (19/40) of patients with IC following OR (P = 0.21). Implementation of our formal rEVAR protocol decreased the incidence of IC significantly from 37.1% (36/97) to 6.4% (10/157), P < 0.001. CONCLUSIONS: The incidence of IC has decreased significantly in the endovascular era but continues to portend a poor prognosis. Implementation of a formal, multidisciplinary rEVAR protocol in our institution decreased the incidence of IC.

Development of the Flu-PRO: a patient-reported outcome (PRO) instrument to evaluate symptoms of influenza.

BACKGROUND: To develop content validity of a comprehensive patient-reported outcome (PRO) measure following current best scientific methodology to standardize assessment of influenza (flu) symptoms in clinical research. METHODS: Stage I (Concept Elicitation): 1:1 telephone interviews with influenza-positive adults (≥18 years) in the US and Mexico within 7 days of diagnosis. Participants described symptom type, character, severity, and duration. Content analysis identified themes and developed the draft Flu-PRO instrument. Stage II (Cognitive Interviewing): The Flu-PRO was administered to a unique set of influenza-positive adults within 14 days of diagnosis; telephone interviews addressed completeness, respondent interpretation of items and ease of use. RESULTS: Samples: Stage I: N = 46 adults (16 US, 30 Mexico); mean (SD) age: 38 (19), 39 (14) years; % female: 56%, 73%; race: 69% White, 97% Mestizo. Stage II: N = 34 adults (12 US, 22 Mexico); age: 37 (14), 39 (11) years; % female: 50%, 50%; race: 58% White, 100% Mestizo. SYMPTOMS: Symptoms identified by >50%: coughing, weak or tired, throat symptoms, congestion, headache, weakness, sweating, chills, general discomfort, runny nose, chest (trouble breathing), difficulty sleeping, and body aches or pains. No new content was uncovered during Stage II; participants easily understood the instrument. CONCLUSIONS: Results show the 37-item Flu-PRO is a content valid measure of influenza symptoms in adults with a confirmed diagnosis of influenza. Research is underway to evaluate the suitability of the instrument for children and adolescents. This work can form the basis for future quantitative tests of reliability, validity, and responsiveness to evaluate the measurement properties of Flu-PRO for use in clinical trials and epidemiology studies.

Modeling bi-modality improves characterization of cell cycle on gene expression in single cells.

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%-17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ≈ 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

Pseudopropionibacterium propionicum as a Cause of Empyema; A Diagnosis with Next-Generation Sequencing.

Pseudopropionibacterium propionicum (P.p.) is an anaerobic, Gram-positive, branching beaded rod that is a component of the human microbiome. An infection of the thoracic cavity with P.p. can mimic tuberculosis (TB), nocardiosis, and malignancy. We present a case of a 77-year-old male who presented with dyspnea and a productive cough who was initially misdiagnosed with TB based on positive acid-fast staining of a pleural biopsy specimen and an elevated adenosine deaminase level of the pleural fluid. He was then diagnosed with nocardiosis based on the Gram stain of his pleural fluid that showed a Gram-positive beaded and branching rod. The pleural fluid specimen was culture-negative, but the diagnosis of thoracic P.p. infection was determined with next-generation sequencing (NGS). The patient was initially treated with imipenem and minocycline, then ceftriaxone and minocycline, and later changed to minocycline only. This report shows the utility of NGS in making a microbiological diagnosis when other techniques either failed to provide a result (culture) or gave misleading information (histopathologic exam, pleural fluid adenosine deaminase determination, and organism morphology on Gram stain).

An information theoretic approach to detecting spatially varying genes.

A key step in spatial transcriptomics is identifying genes with spatially varying expression patterns. We adopt an information theoretic perspective to this problem by equating the degree of spatial coherence with the Jensen-Shannon divergence between pairs of nearby cells and pairs of distant cells. To avoid the notoriously difficult problem of estimating information theoretic divergences, we use modern approximation techniques to implement a computationally efficient algorithm designed to scale with in situ spatial transcriptomics technologies. In addition to being highly scalable, we show that our method, which we call maximization of spatial information (Maxspin), improves accuracy across several spatial transcriptomics platforms and a variety of simulations when compared with a variety of state-of-the-art methods. To further demonstrate the method, we generated in situ spatial transcriptomics data in a renal cell carcinoma sample using the CosMx Spatial Molecular Imager and used Maxspin to reveal novel spatial patterns of tumor cell gene expression.

Eczema Monkeypoxicum in a Female Patient With Atopic Dermatitis.

A female patient with atopic dermatitis who had recently received a tattoo presented with severe right ear pain and several vesiculopustular lesions. Over 1 week, she developed approximately 80 widely distributed lesions. Laboratory testing confirmed mpox (previously monkeypox) virus, and no further lesions developed after initiation of oral tecovirimat.

Single cell spatial transcriptomic profiling of childhood-onset lupus nephritis reveals complex interactions between kidney stroma and infiltrating immune cells.

Children with systemic lupus erythematosus (SLE) are at increased risk of developing kidney disease, termed childhood-onset lupus nephritis (cLN). Single cell transcriptomics of dissociated kidney tissue has advanced our understanding of LN pathogenesis, but loss of spatial resolution prevents interrogation of in situ cellular interactions. Using a technical advance in spatial transcriptomics, we generated a spatially resolved, single cell resolution atlas of kidney tissue (>400,000 cells) from eight cLN patients and two controls. Annotated cells were assigned to 35 reference cell types, including major kidney subsets and infiltrating immune cells. Analysis of spatial distribution demonstrated that individual immune lineages localize to specific regions in cLN kidneys, including myeloid cells trafficking to inflamed glomeruli and B cells clustering within tubulointerstitial immune hotspots. Notably, gene expression varied as a function of tissue location, demonstrating how incorporation of spatial data can provide new insights into the immunopathogenesis of SLE. Alterations in immune phenotypes were accompanied by parallel changes in gene expression by resident kidney stromal cells. However, there was little correlation between histologic scoring of cLN disease activity and glomerular cell transcriptional signatures at the level of individual glomeruli. Finally, we identified modules of spatially-correlated gene expression with predicted roles in induction of inflammation and the development of tubulointerstitial fibrosis. In summary, single cell spatial transcriptomics allows unprecedented insights into the molecular heterogeneity of cLN, paving the way towards more targeted and personalized treatment approaches.

Advances in mixed cell deconvolution enable quantification of cell types in spatial transcriptomic data.

Mapping cell types across a tissue is a central concern of spatial biology, but cell type abundance is difficult to extract from spatial gene expression data. We introduce SpatialDecon, an algorithm for quantifying cell populations defined by single cell sequencing within the regions of spatial gene expression studies. SpatialDecon incorporates several advancements in gene expression deconvolution. We propose an algorithm harnessing log-normal regression and modelling background, outperforming classical least-squares methods. We compile cell profile matrices for 75 tissue types. We identify genes whose minimal expression by cancer cells makes them suitable for immune deconvolution in tumors. Using lung tumors, we create a dataset for benchmarking deconvolution methods against marker proteins. SpatialDecon is a simple and flexible tool for mapping cell types in spatial gene expression studies. It obtains cell abundance estimates that are spatially resolved, granular, and paired with highly multiplexed gene expression data.

Respiratory Infections Are More Common Than Healthcare Records Indicate: Results From an Anonymous Survey.

INTRODUCTION: Influenza-like illnesses (ILIs) are common in military populations and can impair mission-readiness, particularly in the current severe acute respiratory syndrome coronavirus 2 pandemic; therefore, it is important to identify potential risk factors for infection and better understand the burden of infection. MATERIALS AND METHODS: A survey was administered to military medical trainees living in a congregated setting on JBSA Fort Sam Houston, Texas, from January 2017 to February 2019. The survey included questions about ILI experience and potential ILI risk factors. RESULTS: 2,121 individuals completed the survey. Respondents had a median age of 21 years, 46% were female, 32.6% were Air Force, 33.6% were Army, and 33.8% were Navy/Marines. Among the 815 (38%) who reported an ILI during training, 40% sought health care. The primary reasons for seeking healthcare included illness severity, concern about transmission, and accessibility of healthcare. Over half (54%) of the trainees who reported an ILI said the ILI had an impact on their performance, including reduced study time, missed physical training, and missed class. Multivariate model results indicate that women and younger trainees (<30 years) were more likely to report having had an ILI (women: OR 1.58, (95% CI 1.30, 1.92); age <30 years: OR 1.58, (1.06, 2.36)). In a subset analysis, those who reported washing their hands 10+ times per day were less likely to report an ILI (OR 0.61 (0.42, 0.89)). CONCLUSIONS: ILIs are likely to be more common during training than healthcare records indicate and may result in decreased training effectiveness. Increasing access to handwashing facilities and education about the importance of handwashing to prevent the spread of disease will likely reduce the ILI burden in this population.

Breath Biomarkers of Influenza Infection.

Background: Volatile organic compounds (VOCs) are produced systemically due to varied physiological states such as oxidative stress and are excreted through the lungs. Benchtop and preliminary clinical data suggest that breath testing may be a useful diagnostic modality for viral respiratory tract infections. Methods: Patients with influenza-like illness (ILI) presenting to a single clinic in San Antonio, Texas, from 3/2017 to 3/2019 submitted a 2-minute breath sample in addition to a nasopharyngeal swab collected for polymerase chain reaction (PCR) assay for respiratory pathogens. VOCs were assayed with gas chromatography-mass spectrometry (GC-MS), and data were analyzed to identify breath VOC biomarkers that discriminated between ILI patients with and without a polymerase chain reaction (PCR) assay that was positive for influenza. Results: Demographic, clinical, PCR, and breath data were available for 237 episodes of ILI, among which 32 episodes (13.5%) were PCR positive for influenza. Twenty candidate VOCs identified patients with influenza with greater than random accuracy. A predictive algorithm using 4 candidate biomarkers identified this group with 78% accuracy (74% sensitivity, 70% specificity). Based on their mass spectra, most of these biomarkers were n-alkane derivatives, consistent with products of oxidative stress. Conclusions: A breath test for VOC biomarkers accurately identified ILI patients with PCR-proven influenza. These findings bolster those of others that a rapid, accurate, universal point-of-care influenza diagnostic test based on assay of exhaled-breath VOCs may be feasible. The next step will be a study of patients with ILI using a simplified method of breath collection that would facilitate translation for use in clinical practice.

Adaptive immune signature in HER2-positive breast cancer in NCCTG (Alliance) N9831 and NeoALTTO trials.

Trastuzumab acts in part through the adaptive immune system. Previous studies showed that enrichment of immune-related gene expression was associated with improved outcomes in HER2-positive (HER2+) breast cancer. However, the role of the immune system in response to lapatinib is not fully understood. Gene expression analysis was performed in 1,268 samples from the North Central Cancer Treatment Group (NCCTG) N9831 and 244 samples from the NeoALTTO trial. In N9831, enrichment of CD45 and immune-subset signatures were significantly associated with improved outcomes. We identified a novel 17-gene adaptive immune signature (AIS), which was found to be significantly associated with improved RFS among patients who received adjuvant trastuzumab (HR 0.66, 95% CI 0.49-0.90, Cox regression model p = 0.01) but not in patients who received chemotherapy alone (HR 0.96, 95% CI 0.67-1.40, Cox regression model p = 0.97). This result was validated in NeoALTTO. Overall, AIS-low patients had a significantly lower pathologic complete response (pCR) rate compared with AIS-high patients (χ2 p < 0.0001). Among patients who received trastuzumab alone, pCR was observed in 41.7% of AIS-high patients compared with 9.8% in AIS-low patients (OR of 6.61, 95% CI 2.09-25.59, logistic regression model p = 0.003). More importantly, AIS-low patients had a higher pCR rate with an addition of lapatinib (51.1% vs. 9.8%, OR 9.65, 95% CI 3.24-36.09, logistic regression model p < 0.001). AIS-low patients had poor outcomes, despite receiving adjuvant trastuzumab. However, these patients appear to benefit from an addition of lapatinib. Further studies are needed to validate the significance of this signature to identify patients who are more likely to benefit from dual anti-HER2 therapy. ClinicalTrials.gov Identifiers: NCT00005970 (NCCTG N9831) and NCT00553358 (NeoALTTO).

Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Aeromonas hydrophila septic arthritis.

Septic arthritis is a serious, life and limb threatening infection. If suspected, empiric treatment must begin immediately and account for the most likely pathogens. Eight days following left knee arthroscopic surgery, a 51-year-old active duty male spent approximately 1 hour driving a personal watercraft on Okaloosa Bay near the Gulf of Mexico. Eight days later, he presented to the emergency room with septic arthritis of that knee. Fluid aspirated from the joint yielded Aeromonas hydrophila. The infection resolved with surgical drainage and 21 days of levofloxacin. A. hydrophila is a rare cause of septic arthritis, and reported cases have involved exposure to water after trauma to the affected joint. Many U.S. military bases are located in coastal areas and military members frequently participate in activities which compromise skin integrity and place them at increased risk for contracting waterborne infections. We present the ninth case of A. hydrophila septic arthritis described in the English language literature, highlight the importance of considering this pathogen in at-risk populations, and review the diagnosis and management of septic arthritis.