Integration of peripheral blood- and tissue-based biomarkers of response to immune checkpoint blockade in urothelial carcinoma.

As predictive biomarkers of response to immune checkpoint inhibitors (ICIs) remain a major unmet clinical need in patients with urothelial carcinoma (UC), we sought to identify tissue-based immune biomarkers of clinical benefit to ICIs using multiplex immunofluorescence and to integrate these findings with previously identified peripheral blood biomarkers of response. Fifty-five pretreatment and 12 paired on-treatment UC specimens were identified from patients treated with nivolumab with or without ipilimumab. Whole tissue sections were stained with a 12-plex mIF panel, including CD8, PD-1/CD279, PD-L1/CD274, CD68, CD3, CD4, FoxP3, TCF1/7, Ki67, LAG-3, MHC-II/HLA-DR, and pancytokeratin+SOX10 to identify over three million cells. Immune tissue densities were compared to progression-free survival (PFS) and best overall response (BOR) by RECIST version 1.1. Correlation coefficients were calculated between tissue-based and circulating immune populations. The frequency of intratumoral CD3+ LAG-3+ cells was higher in responders compared to nonresponders (p = 0.0001). LAG-3+ cellular aggregates were associated with response, including CD3+ LAG-3+ in proximity to CD3+ (p = 0.01). Exploratory multivariate modeling showed an association between intratumoral CD3+ LAG-3+ cells and improved PFS independent of prognostic clinical factors (log HR -7.0; 95% confidence interval [CI] -12.7 to -1.4), as well as established biomarkers predictive of ICI response (log HR -5.0; 95% CI -9.8 to -0.2). Intratumoral LAG-3+ immune cell populations warrant further study as a predictive biomarker of clinical benefit to ICIs. Differences in LAG-3+ lymphocyte populations across the intratumoral and peripheral compartments may provide complementary information that could inform the future development of multimodal composite biomarkers of ICI response. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Perioperative Methadone and Ketamine for Postoperative Pain Control in Spinal Surgical Patients: A Randomized, Double-blind, Placebo-controlled Trial.

BACKGROUND: Despite application of multimodal pain management strategies, patients undergoing spinal fusion surgery frequently report severe postoperative pain. Methadone and ketamine, which are N-methyl-d-aspartate receptor antagonists, have been documented to facilitate postoperative pain control. This study therefore tested the primary hypothesis that patients recovering from spinal fusion surgery who are given ketamine and methadone use less hydromorphone on the first postoperative day than those give methadone alone. METHODS: In this randomized, double-blind, placebo-controlled trial, 130 spinal surgery patients were randomized to receive either methadone at 0.2 mg/kg (ideal body weight) intraoperatively and a 5% dextrose in water infusion for 48 h postoperatively (methadone group) or 0.2 mg/kg methadone intraoperatively and a ketamine infusion (0.3 mg · kg-1 · h-1 infusion [no bolus] intraoperatively and then 0.1 mg · kg-1 · h-1 for next 48 h [both medications dosed at ideal body weight]; methadone/ketamine group). Anesthetic care was standardized in all patients. Intravenous hydromorphone use on postoperative day 1 was the primary outcome. Pain scores, intravenous and oral opioid requirements, and patient satisfaction with pain management were assessed for the first 3 postoperative days. RESULTS: Median (interquartile range) intravenous hydromorphone requirements were lower in the methadone/ketamine group on postoperative day 1 (2.0 [1.0 to 3.0] vs. 4.6 [3.2 to 6.6] mg in the methadone group, median difference [95% CI] 2.5 [1.8 to 3.3] mg; P < 0.0001) and postoperative day 2. In addition, fewer oral opioid tablets were needed in the methadone/ketamine group on postoperative day 1 (2 [0 to 3] vs. 4 [0 to 8] in the methadone group; P = 0.001) and postoperative day 3. Pain scores at rest, with coughing, and with movement were lower in the methadone/ketamine group at 23 of the 24 assessment times. Patient-reported satisfaction scores were high in both study groups. CONCLUSIONS: Postoperative analgesia was enhanced by the combination of methadone and ketamine, which act on both N-methyl-d-aspartate and µ-opioid receptors. The combination could be considered in patients having spine surgery.

Neuromuscular and Clinical Recovery in Thoracic Surgical Patients Reversed With Neostigmine or Sugammadex.

BACKGROUND: Patients undergoing thoracoscopic procedures may be at high-risk for incomplete neuromuscular recovery and associated complications. The aim of this clinical investigation was to assess the incidence of postoperative residual neuromuscular blockade in adult thoracic surgical patients administered neostigmine or sugammadex when optimal dosing and reversal strategies for these agents were used. The effect of choice of reversal agent on hypoxemic events and signs and symptoms of muscle weakness were also determined. Additionally, operative conditions in each group were graded by surgeons performing the procedures. METHODS: Two hundred patients undergoing thoracoscopic surgical procedures were enrolled in this nonrandomized controlled trial. One hundred consecutive patients maintained at moderate levels of neuromuscular blockade were reversed with neostigmine (neostigmine group) followed by 100 consecutive patients given sugammadex to antagonize deeper levels of neuromuscular blockade (sugammadex group). Anesthetic and neuromuscular management were standardized. Surgeons rated operative conditions at the conclusion of the procedure on a 4-point scale (grade 1 = excellent to grade 4 = poor). Train-of-four ratios were measured immediately before extubation and at PACU admission (primary outcomes). Postoperatively, patients were assessed for adverse respiratory events and 11 signs and 16 symptoms of muscle weakness. RESULTS: The 2 groups were similar in intraoperative management characteristics. The percentage of patients with residual neuromuscular blockade, defined as a normalized train-of-four ratio <0.9, was significantly greater in the neostigmine group than the sugammadex group at both tracheal extubation (80% vs 6%, respectively, P < .0001) and PACU admission (61% vs 1%, respectively, P < .0001). Patients in the neostigmine group had less optimal operative conditions (median score 2 [good] versus 1 [excellent] in the sugammadex group; P < .0001), and more symptoms of muscle weakness were present in these subjects (median number [interquartile range] 4 [1-8] vs 1 [0-2] in the sugammadex group, P < .0001). No differences between groups in adverse airway events were observed. CONCLUSIONS: Despite the application of strategies documented to reduce the risk of residual neuromuscular blockade, a high percentage of thoracoscopic patients whose neuromuscular blockade was reversed with neostigmine were admitted to the PACU with clinical evidence of residual paralysis. In contrast, muscle weakness was rarely observed in patients whose neuromuscular blockade was antagonized with sugammadex.

Postoperative Pain and Analgesic Requirements in the First Year after Intraoperative Methadone for Complex Spine and Cardiac Surgery.

BACKGROUND: Methadone is a long-acting opioid that has been reported to reduce postoperative pain scores and analgesic requirements and may attenuate development of chronic postsurgical pain. The aim of this secondary analysis of two previous trials was to follow up with patients who had received a single intraoperative dose of either methadone or traditional opioids for complex spine or cardiac surgical procedures. METHODS: Preplanned analyses of long-term outcomes were conducted for spinal surgery patients randomized to receive 0.2 mg/kg methadone at the start of surgery or 2 mg hydromorphone at surgical closure, and for cardiac surgery patients randomized to receive 0.3 mg/kg methadone or 12 µg/kg fentanyl intraoperatively. A pain questionnaire assessing the weekly frequency (the primary outcome) and intensity of pain was mailed to subjects 1, 3, 6, and 12 months after surgery. Ordinal data were compared with the Mann-Whitney U test, and nominal data were compared using the chi-square test or Fisher exact probability test. The criterion for rejection of the null hypothesis was P < 0.01. RESULTS: Three months after surgery, patients randomized to receive methadone for spine procedures reported the weekly frequency of chronic pain was less (median score 0 on a 0 to 4 scale [less than once a week] vs. 3 [daily] in the hydromorphone group, P = 0.004). Patients randomized to receive methadone for cardiac surgery reported the frequency of postsurgical pain was less at 1 month (median score 0) than it was in patients randomized to receive fentanyl (median score 2 [twice per week], P = 0.004). CONCLUSIONS: Analgesic benefits of a single dose of intraoperative methadone were observed during the first 3 months after spinal surgery (but not at 6 and 12 months), and during the first month after cardiac surgery, when the intensity and frequency of pain were the greatest.

Neostigmine Administration after Spontaneous Recovery to a Train-of-Four Ratio of 0.9 to 1.0: A Randomized Controlled Trial of the Effect on Neuromuscular and Clinical Recovery.

BACKGROUND: When a muscle relaxant is administered to facilitate intubation, the benefits of anticholinesterase reversal must be balanced with potential risks. The aim of this double-blinded, randomized noninferiority trial was to evaluate the effect of neostigmine administration on neuromuscular function when given to patients after spontaneous recovery to a train-of-four ratio of 0.9 or greater. METHODS: A total of 120 patients presenting for surgery requiring intubation were given a small dose of rocuronium. At the conclusion of surgery, 90 patients achieving a train-of-four ratio of 0.9 or greater were randomized to receive either neostigmine 40 µg/kg or saline (control). Train-of-four ratios were measured from the time of reversal until postanesthesia care unit admission. Patients were monitored for postextubation adverse respiratory events and assessed for muscle strength. RESULTS: Ninety patients achieved a train-of-four ratio of 0.9 or greater at the time of reversal. Mean train-of-four ratios in the control and neostigmine groups before reversal (1.02 vs. 1.03), 5 min postreversal (1.05 vs. 1.07), and at postanesthesia care unit admission (1.06 vs. 1.08) did not differ. The mean difference and corresponding 95% CI of the latter were -0.018 and -0.046 to 0.010. The incidences of postoperative hypoxemic events and episodes of airway obstruction were similar for the groups. The number of patients with postoperative signs and symptoms of muscle weakness did not differ between groups (except for double vision: 13 in the control group and 2 in the neostigmine group; P = 0.001). CONCLUSIONS: Administration of neostigmine at neuromuscular recovery was not associated with clinical evidence of anticholinesterase-induced muscle weakness. VISUAL ABSTRACT: An online visual overview is available for this article.(Figure is included in full-text article.).

Comparison of the TOFscan and the TOF-Watch SX during Recovery of Neuromuscular Function.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Quantitative neuromuscular monitoring is required to ensure neuromuscular function has recovered completely at the time of tracheal extubation. The TOFscan (Drager Technologies, Canada) is a new three-dimensional acceleromyography device that measures movement of the thumb in multiple planes. The aim of this observational investigation was to assess the agreement between nonnormalized and normalized train-of-four values obtained with the TOF-Watch SX (Organon, Ireland) and those obtained with the TOFscan during recovery from neuromuscular blockade. METHODS: Twenty-five patients were administered rocuronium, and spontaneous recovery of neuromuscular blockade was allowed to occur. The TOFscan and TOF-Watch SX devices were applied to opposite arms. A preload was applied to the TOF-Watch SX, and calibration was performed before rocuronium administration. Both devices were activated, and train-of-four values were obtained every 15 s. Modified Bland-Altman analyses were conducted to compare train-of-four ratios measured with the TOFscan to those measured with the TOF-Watch SX (when train-of-four thresholds of 0.2 to 1.0 were achieved). RESULTS: Bias and 95% limits of agreement between the TOF-Watch SX and the TOFscan at nonnormalized train-of-four ratios between 0.2 and 1.0 were 0.021 and -0.100 to 0.141, respectively. When train-of-four measures with the TOF-Watch SX were normalized, bias and 95% limits of agreement between the TOF-Watch SX and the TOFscan at ratios between 0.2 and 1.0 were 0.015 and -0.097 to 0.126, respectively. CONCLUSIONS: Good agreement between the TOF-Watch SX with calibration and preload application and the uncalibrated TOFscan was observed throughout all stages of neuromuscular recovery.

Impact of an Activity-Based Program on Health, Quality of Life, and Occupational Performance of Women Diagnosed With Cancer.

OBJECTIVE: We evaluated the impact of a 1-wk activity program on the health, quality of life (QOL), and occupational performance of community-living women diagnosed with cancer. METHOD: A one-group pretest-posttest repeated-measures design was used. Participants completed a functional health measure (36-Item Short Form Health Survey [SF-36]), a QOL measure (World Health Organization Quality of Life-Brief version [WHOQOL-BREF]), and an occupational performance and satisfaction measure (Canadian Occupational Performance Measure [COPM]) before and 6 wk after program completion. The COPM was also administered on Day 5. RESULTS: Paired t tests for the SF-36 and WHOQOL-BREF showed no significant differences, except for the WHOQOL-BREF's Social Relationships subscale (p < .008). Repeated-measures analyses of variance showed a significant difference in COPM performance and satisfaction scores (p < .001). CONCLUSION: The activity program effectively improved occupational performance and satisfaction and social relationships of community-living women diagnosed with cancer.

Early on-treatment assessment of T-cells, cytokines and tumor DNA with adaptively-dosed nivolumab + ipilimumab: final results from the phase 2 ADAPT-IT study.

PURPOSE: ADAPT-IT (NCT03122522) investigated adaptive ipilimumab discontinuation in melanoma based on early radiographic assessment. Initial findings indicated similar effectiveness compared to conventional nivolumab-ipilimumab (nivo-ipi). Exploratory biomarker analyses and final clinical results are now reported. PATIENTS AND METHODS: Patients with unresectable melanoma received two doses of nivo-ipi. Radiographic assessment at Week 6 informed continuation of ipilimumab before nivolumab maintenance. The primary endpoint was overall response rate (ORR) at Week 12. Plasma was assayed for circulating tumor DNA (ctDNA) and ten cytokines using a multiplex immunoassay. Flow cytometry of peripheral blood mononuclear cells was performed with an 11-color panel. RESULTS: Among treated patients, expansion of proliferating T-cell populations was observed in both responders and non-responders. Baseline IL-6 levels were lower in patients achieving an objective radiographic response (median 1.30 vs 2.86 pg/mL; p=0.025). Higher baseline IL-6 levels were associated with shorter progression-freesurvival (PFS; hazard ratio (HR)=1.24, 95% CI:1.01-1.52; p=0.041). At Week 6, patients with response had lower average tumor variant allele fractions (VAF) compared to non-responders (median 0.000 v 0.019; p=0.014). Greater increases in average VAF from baseline to Week 6 correlated with shorter PFS (HR=1.11, 95% CI:1.01-1.21; p=0.023). Week 12 ORR was 47% (95% CI:35-59%) with a median follow-up of 34 months among survivors. Median PFS was 21 months (95% CI:10-not reached); 76% of responses (95% CI:64%-91%) persisted at 36 months. CONCLUSIONS: Adaptively dosed nivo-ipi responses are durable and resemble historical data for conventional nivo-ipi. Baseline IL-6 and ctDNA changes during treatment warrant further study as biomarkers of nivo-ipi response.

Ultrasensitive plasma-based monitoring of tumor burden using machine-learning-guided signal enrichment.

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.

Uncovering the hidden structure of dynamic T cell composition in peripheral blood during cancer immunotherapy: a topic modeling approach.

Immune checkpoint inhibitors (ICIs), now mainstays in the treatment of cancer treatment, show great potential but only benefit a subset of patients. A more complete understanding of the immunological mechanisms and pharmacodynamics of ICI in cancer patients will help identify the patients most likely to benefit and will generate knowledge for the development of next-generation ICI regimens. We set out to interrogate the early temporal evolution of T cell populations from longitudinal single-cell flow cytometry data. We developed an innovative statistical and computational approach using a Latent Dirichlet Allocation (LDA) model that extends the concept of topic modeling used in text mining. This powerful unsupervised learning tool allows us to discover compositional topics within immune cell populations that have distinct functional and differentiation states and are biologically and clinically relevant. To illustrate the model's utility, we analyzed ∼17 million T cells obtained from 138 pre- and on-treatment peripheral blood samples from a cohort of melanoma patients treated with ICIs. We identified three latent dynamic topics: a T-cell exhaustion topic that recapitulates a LAG3+ predominant patient subgroup with poor clinical outcome; a naive topic that shows association with immune-related toxicity; and an immune activation topic that emerges upon ICI treatment. We identified that a patient subgroup with a high baseline of the naïve topic has a higher toxicity grade. While the current application is demonstrated using flow cytometry data, our approach has broader utility and creates a new direction for translating single-cell data into biological and clinical insights.

A topic modeling approach reveals the dynamic T cell composition of peripheral blood during cancer immunotherapy.

We present TopicFlow, a computational framework for flow cytometry data analysis of patient blood samples for the identification of functional and dynamic topics in circulating T cell population. This framework applies a Latent Dirichlet Allocation (LDA) model, adapting the concept of topic modeling in text mining to flow cytometry. To demonstrate the utility of our method, we conducted an analysis of ∼17 million T cells collected from 138 peripheral blood samples in 51 patients with melanoma undergoing treatment with immune checkpoint inhibitors (ICIs). Our study highlights three latent dynamic topics identified by LDA: a T cell exhaustion topic that independently recapitulates the previously identified LAG-3+ immunotype associated with ICI resistance, a naive topic and its association with immune-related toxicity, and a T cell activation topic that emerges upon ICI treatment. Our approach can be broadly applied to mine high-parameter flow cytometry data for insights into mechanisms of treatment response and toxicity.

Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma.

CD8+ T cell reactivity towards tumor mutation-derived neoantigens is widely believed to facilitate the antitumor immunity induced by immune checkpoint blockade (ICB). Here we show that broadening in the number of neoantigen-reactive CD8+ T cell (NART) populations between pre-treatment to 3-weeks post-treatment distinguishes patients with controlled disease compared to patients with progressive disease in metastatic urothelial carcinoma (mUC) treated with PD-L1-blockade. The longitudinal analysis of peripheral CD8+ T cell recognition of patient-specific neopeptide libraries consisting of DNA barcode-labelled pMHC multimers in a cohort of 24 patients from the clinical trial NCT02108652 also shows that peripheral NARTs derived from patients with disease control are characterised by a PD1+ Ki67+ effector phenotype and increased CD39 levels compared to bystander bulk- and virus-antigen reactive CD8+ T cells. The study provides insights into NART characteristics following ICB and suggests that early-stage NART expansion and activation are associated with response to ICB in patients with mUC.

Checkpoint Blockade in Melanoma Patients With Underlying Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is associated with immune dysfunction and an increased risk of melanoma. For patients with metastatic melanoma, immunotherapy with checkpoint blocking antibodies is a standard of care. In patients with concomitant CLL and metastatic melanoma, it is not known whether CLL might influence the antimelanoma efficacy or immune-related toxicities of immune checkpoint blockade. Fifteen patients with locally advanced or metastatic melanoma and a concomitant diagnosis of CLL who received pembrolizumab or ipilimumab with or without nivolumab for the treatment of their melanoma at Memorial Sloan Kettering Cancer Center between January 1, 2010, and January 1, 2017, were retrospectively identified. Clinical characteristics including absolute lymphocyte counts during therapy were recorded along with a response to treatment (objective radiographic response, progression-free survival, and adverse events) for each patient. Of 9 response-evaluable patients treated with ipilimumab, 3 (33%) had a partial response, 1 (11%) had stable disease, and 5 (56%) developed progressive disease. Objective tumor responses were also observed with single-agent therapy pembrolizumab and with combination therapy of nivolumab and ipilimumab. Grade 3 or 4 toxicity was observed in 6 of 15 patients (40%), including diarrhea, transaminitis, rash, and hemolytic anemia. Although our retrospective assessment was limited, there was no evidence that CLL responded to the checkpoint blockade. This case series demonstrates that ipilimumab, pembrolizumab, and combined ipilimumab and nivolumab therapies show clinical activity in patients with melanoma and concomitant CLL, at rates consistent with those previously reported. This population may warrant closer surveillance for hematologic immune-related toxicities such as autoimmune hemolytic anemia.

LAG-3 expression on peripheral blood cells identifies patients with poorer outcomes after immune checkpoint blockade.

Immune checkpoint blocking antibodies are a cornerstone in cancer treatment; however, they benefit only a subset of patients and biomarkers to guide immune checkpoint blockade (ICB) treatment choices are lacking. We designed this study to identify blood-based correlates of clinical outcome in ICB-treated patients. We performed immune profiling of 188 ICB-treated patients with melanoma using multiparametric flow cytometry to characterize immune cells in pretreatment peripheral blood. A supervised statistical learning approach was applied to a discovery cohort to classify phenotypes and determine their association with survival and treatment response. We identified three distinct immune phenotypes (immunotypes), defined in part by the presence of a LAG-3+CD8+ T cell population. Patients with melanoma with a LAG+ immunotype had poorer outcomes after ICB with a median survival of 22.2 months compared to 75.8 months for those with the LAG- immunotype (P = 0.031). An independent cohort of 94 ICB-treated patients with urothelial carcinoma was used for validation where LAG+ immunotype was significantly associated with response (P = 0.007), survival (P < 0.001), and progression-free survival (P = 0.004). Multivariate Cox regression and stratified analyses further showed that the LAG+ immunotype was an independent marker of outcome when compared to known clinical prognostic markers and previously described markers for the clinical activity of ICB, PD-L1, and tumor mutation burden. The pretreatment peripheral blood LAG+ immunotype detects patients who are less likely to benefit from ICB and suggests a strategy for identifying actionable immune targets for further investigation.