Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma.

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.

High-dose IV magnesium in mesothelioma patients receiving surgery with hyperthermic intraoperative cisplatin: Pilot studies and design of a phase II randomized clinical trial.

INTRODUCTION: Hyperthermic intraoperative cisplatin (HIOC) is associated with acute kidney injury (AKI). Administration of high-dose magnesium attenuates cisplatin-induced AKI (CP-AKI) in animal models but has not been rigorously examined in humans. METHODS: We tested the feasibility and safety of different doses of magnesium in mesothelioma patients receiving HIOC. In Pilot Study 1, we administered a 36-h continuous infusion of magnesium at 0.5 g/h, targeting serum magnesium levels between 3 and 4.8 mg/dL. In Pilot Study 2A, we administered a 6 g bolus followed by an infusion starting at 2 g/h, titrated to achieve levels between 4 and 6 mg/dL. We eliminated the bolus in Pilot Study 2B. RESULTS: In Pilot Study 1, all five patients enrolled completed the study; however, median postoperative Mg levels were only 2.4 mg/dL. In Pilot Study 2A, two of four patients (50%) were withdrawn due to bradycardia during the bolus. In Pilot Study 2B, two patients completed the study whereas two developed postoperative bradycardia attributed to the magnesium. CONCLUSIONS: A 0.5 g/h infusion for 36 h did not achieve therapeutic magnesium levels, while an infusion at 2 g/h was associated with bradycardia. These studies informed the design of a randomized clinical trial testing whether intravenously Mg attenuates HIOC-associated AKI.

Clinical and molecular validation of BAP1, MTAP, P53, and Merlin immunohistochemistry in diagnosis of pleural mesothelioma.

BAP1 and MTAP immunostains play an important role in diagnosis of mesothelioma, but additional markers are needed to increase sensitivity. We analyzed 84 pleural mesotheliomas (51 epithelioid, 27 biphasic, 6 sarcomatoid) by a hybrid-capture next-generation sequencing (NGS) panel including complete coverage of coding and splicing regions for BAP1, CDKN2A/MTAP, NF2, and TP53 and correlated molecular findings with diagnostic immunostains for BAP1, MTAP, Merlin, and p53, respectively. Fifty-seven reactive mesothelial proliferations served as benign comparators. Loss of BAP1, MTAP, and Merlin protein expression were, respectively, 54%, 46%, and 52% sensitive and 100% specific for mesothelioma. Two-marker immunopanels of BAP1 + MTAP, BAP1 + Merlin, and MTAP + Merlin were 79%, 85%, and 71% sensitive for mesothelioma, while a three-marker immunopanel of BAP1 + MTAP + Merlin was 90% sensitive. Diffuse (mutant-pattern) p53 immunostaining was seen in only 6 (7%) tumors but represented the only immunohistochemical abnormality in 2 cases. Null-pattern p53 was not specific for malignancy. An immunopanel of BAP1 + MTAP + Merlin + p53 was 93% sensitive for mesothelioma, and panel NGS detected a pathogenic alteration in BAP1, MTAP, NF2, and/or TP53 in 95%. Together, 83 (99%) of 84 tumors showed a diagnostic alteration by either immunohistochemistry or panel NGS. Adding Merlin to the standard BAP1 + MTAP immunopanel increases sensitivity for mesothelioma without sacrificing specificity. p53 immunohistochemistry and panel NGS with complete coverage of BAP1, CDKN2A/MTAP, TP53, and NF2 may be useful in diagnostically challenging cases.

Mediastinal Masses, Anesthetic Interventions, and Airway Compression in Adults: A Prospective Observational Study.

BACKGROUND: Central airway occlusion is a feared complication of general anesthesia in patients with mediastinal masses. Maintenance of spontaneous ventilation and avoiding neuromuscular blockade are recommended to reduce this risk. Physiologic arguments supporting these recommendations are controversial and direct evidence is lacking. The authors hypothesized that, in adult patients with moderate to severe mediastinal mass-mediated tracheobronchial compression, anesthetic interventions including positive pressure ventilation and neuromuscular blockade could be instituted without compromising central airway patency. METHODS: Seventeen adult patients with large mediastinal masses requiring general anesthesia underwent awake intubation followed by continuous video bronchoscopy recordings of the compromised portion of the airway during staged induction. Assessments of changes in anterior-posterior airway diameter relative to baseline (awake, spontaneous ventilation) were performed using the following patency scores: unchanged = 0; 25 to 50% larger = +1; more than 50% larger = +2; 25 to 50% smaller = -1; more than 50% smaller = -2. Assessments were made by seven experienced bronchoscopists in side-by-side blinded and scrambled comparisons between (1) baseline awake, spontaneous breathing; (2) anesthetized with spontaneous ventilation; (3) anesthetized with positive pressure ventilation; and (4) anesthetized with positive pressure ventilation and neuromuscular blockade. Tidal volumes, respiratory rate, and inspiratory/expiratory ratio were similar between phases. RESULTS: No significant change from baseline was observed in the mean airway patency scores after the induction of general anesthesia (0 [95% CI, 0 to 0]; P = 0.953). The mean airway patency score increased with the addition of positive pressure ventilation (0 [95% CI, 0 to 1]; P = 0.024) and neuromuscular blockade (1 [95% CI, 0 to 1]; P < 0.001). No patient suffered airway collapse or difficult ventilation during any anesthetic phase. CONCLUSIONS: These observations suggest a need to reassess prevailing assumptions regarding positive pressure ventilation and/or paralysis and mediastinal mass-mediated airway collapse, but do not prove that conventional (nonstaged) inductions are safe for such patients.

Image-guided video-assisted thoracoscopic resection (iVATS): Translation to clinical practice-real-world experience.

OBJECTIVE: We developed a novel approach for localization and resection of lung nodules, using image-guided video-assisted thoracoscopic surgery (iVATS). We report our experience of translating iVATS into clinical care. METHODS: Methodology and workflow for iVATS developed as part of the Phase I/II trial were used to train surgeons, radiologists, anesthesiologists, and radiology technologists. Radiation dose, time from induction to incision, placement of T-bar to incision and incision to closure, hospital stay, and complication rates were recorded. RESULTS: Fifty patients underwent iVATS for resection of 54 nodules in a clinical hybrid operating room (OR) by six surgeons. Fifty-two (97%) nodules were successfully resected. Forty-two (84%) patients underwent wedge resection, four (7%) lobectomies, and two (4%) segmentectomy all with lymph node dissection. Median time from induction to incision was 89 minutes (range: 13-256 minutes); T-bar placement was 14 minutes (10-29 minutes); and incision to closure, 107 minutes (41-302 minutes). Average and total procedure radiation dose were: median = 6 mSieverts (range: 2.9-35 mSieverts). No deaths were reported and median length of stay was 3 days (range: 1-12 days). CONCLUSIONS: Translation of iVATS into clinical practice has been initiated using a safe step-wise process, combining intraoperative C-arm computed tomography scanning and thoracoscopic surgery in a hybrid OR.

Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors.

PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45+ immune cells, a significantly higher proportion of infiltrating CD3+ T cells, and a significantly higher percentage of CD3+ cells displaying the activated HLA-DR+/CD38+ phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8+ T cells, a higher fraction of FOXP3+/CD4+ Tregs, and increased expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells. Double-positive PD-1+/TIM-3+ CD8+ T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3+ T cells and PD-1+/TIM-3+ CD8+ T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR.

Image-guided video assisted thoracoscopic surgery (iVATS) - phase I-II clinical trial.

PURPOSE: To facilitate localization and resection of small lung nodules, we developed a prospective clinical trial (ClinicalTrials.gov number NCT01847209) for a novel surgical approach which combines placement of fiducials using intra-operative C-arm computed tomography (CT) guidance with standard thoracoscopic resection technique using image-guided video-assisted thoracoscopic surgery (iVATS). METHODS: Pretrial training was performed in a porcine model using C-arm CT and needle guidance software. Methodology and workflow for iVATS was developed, and a multi-modality team was trained. A prospective phase I-II clinical trial was initiated with the goal of recruiting eligible patients with small peripheral pulmonary nodules. Intra-operative C-arm CT scan was utilized for guidance of percutaneous marking with two T-bars (Kimberly-Clark, Roswell, GA) followed by VATS resection of the tumor. RESULTS: Twenty-five patients were enrolled; 23 underwent iVATS, one withdrew, and one lesion resolved. Size of lesions were: 0.6-1.8 cm, mean = 1.3 ± 0.38 cm.. All 23 patients underwent complete resection of their lesions. CT imaging of the resected specimens confirmed the removal of the T-bars and the nodule. Average and total procedure radiation dose was in the acceptable low range (median = 1501 µGy*m(2), range 665-16,326). There were no deaths, and all patients were discharged from the hospital (median length of stay = 4 days, range 2-12). Three patients had postoperative complications: one prolonged air-leak, one pneumonia, and one ileus. CONCLUSIONS: A successful and safe step-wise process has been established for iVATS, combining intra-operative C-arm CT scanning and thoracoscopic surgery in a hybrid operating room.