RB constrains lineage fidelity and multiple stages of tumour progression and metastasis.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1-3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4-6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7-9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy.

Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

A clinical trial of intraoperative near-infrared imaging to assess tumor extent and identify residual disease during anterior mediastinal tumor resection.

BACKGROUND: The management of most solid tumors of the anterior mediastinum involves complete resection. Because of their location near mediastinal structures, wide resection is not possible; therefore, surgeons must use subjective visual and tactile cues to determine disease extent. This clinical trial explored intraoperative near-infrared (NIR) imaging as an approach to improving tumor delineation during mediastinal tumor resection. METHODS: Twenty-five subjects with anterior mediastinal lesions suspicious for malignancy were enrolled in an open-label feasibility trial. Subjects were administered indocyanine green (ICG) at a dose of 5 mg/kg, 24 hours before resection (via a technique called TumorGlow). The NIR imaging systems included Artemis (Quest, Middenmeer, the Netherlands) and Iridium (VisionSense Corp, Philadelphia, Pennsylvania). Intratumoral ICG uptake was evaluated. The clinical value was determined via an assessment of the ability of NIR imaging to detect phrenic nerve involvement or incomplete resection. Clinical and histopathologic variables were analyzed to determine predictors of tumor fluorescence. RESULTS: No drug-related toxicity was observed. Optical imaging added a mean of 10 minutes to case duration. Among the subjects with solid tumors, 19 of 20 accumulated ICG. Fluorescent tumors included thymomas (n = 13), thymic carcinomas (n = 4), and liposarcomas (n = 2). NIR feedback improved phrenic nerve dissection (n = 4) and identified residual disease (n = 2). There were no false-positives or false-negatives. ICG preferentially accumulated in solid tumors; this was independent of clinical and pathologic variables. CONCLUSIONS: NIR imaging for anterior mediastinal neoplasms is safe and feasible. This technology may provide a real-time tool capable of determining tumor extent and specifically identify phrenic nerve involvement and residual disease.

Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas.

Non-small cell lung cancer (NSCLC) is the number one cancer killer in the United States. Despite attempted curative surgical resection, nearly 40% of patients succumb to recurrent disease. High recurrence rates may be partially explained by data suggesting that 20% of NSCLC patients harbor synchronous disease that is missed during resection. In this report, we describe the use of a novel folate receptor-targeted near-infrared contrast agent (OTL38) to improve the intraoperative localization of NSCLC during pulmonary resection. Using optical phantoms, fluorescent imaging with OTL38 was associated with less autofluorescence and greater depth of detection compared to traditional optical contrast agents. Next, in in vitro and in vivo NSCLC models, OTL38 reliably localized NSCLC models in a folate receptor-dependent manner. Before testing intraoperative molecular imaging with OTL38 in humans, folate receptor-alpha expression was confirmed to be present in 86% of pulmonary adenocarcinomas upon histopathologic review of 100 human pulmonary resection specimens. Lastly, in a human feasibility study, intraoperative molecular imaging with OTL38 accurately identified 100% of pulmonary adenocarcinomas and allowed for identification of additional subcentimeter neoplastic processes in 30% of subjects. This technology may enhance the surgeon's ability to identify NSCLC during oncologic resection and potentially improve long-term outcomes.

Intraoperative Molecular Imaging Combined With Positron Emission Tomography Improves Surgical Management of Peripheral Malignant Pulmonary Nodules.

OBJECTIVE: To determine if intraoperative molecular imaging (IMI) can improve detection of malignant pulmonary nodules. BACKGROUND: 18-Fluorodeoxyglucose positron emission tomography (PET) is commonly utilized in preoperative assessment of patients with solid malignancies; however, false negatives and false positives remain major limitations. Using patients with pulmonary nodules as a study model, we hypothesized that IMI with a folate receptor targeted near-infrared contrast agent (OTL38) can improve malignant pulmonary nodule identification when combined with PET. METHODS: Fifty patients with pulmonary nodules with imaging features suspicious for malignancy underwent preoperative PET. Patients then received OTL38 before pulmonary resection. During resection, IMI was utilized to evaluate known pulmonary nodules and identify synchronous lesions. Tumor size, PET standardized uptake value, and IMI tumor-to-background ratios were compared for known and synchronous nodules via paired and unpaired t tests, when appropriate. Test characteristics of PET and IMI with OTL38 were compared. RESULTS: IMI identified 56 of 59 (94.9%) malignant pulmonary nodules identified by preoperative imaging. IMI located an additional 9 malignant lesions not identified preoperatively. Nodules only detected by IMI were smaller than nodules detected preoperatively (0.5 vs 2.4 cm; P < 0.01), but displayed similar fluorescence (tumor-to-background ratio 3.3 and 3.1; P = 0.50). Sensitivity of IMI and PET were 95.6% and 73.5% (P = 0.001), respectively; and positive predictive values were 94.2% and 89.3%, respectively (P > 0.05). Additionally, utilization of IMI clinically upstaged 6 (12%) subjects and improved management of 15 (30%) subjects. CONCLUSIONS: These data suggest that combining IMI with PET may provide superior oncologic outcomes for patients with resectable lung cancer.

Bilateral Lung Transplantation for Bleomycin-Associated Lung Injury.

This report details the successful use of bilateral lung transplantation for the management of severe postoperative bleomycin-associated lung injury. This case highlights that the extremely favorable prognosis of advanced testicular germ cell tumors after systemic chemotherapy (>90% cure rate) should not preclude lung transplant consideration in all cases, despite current guidance that considers an advanced malignancy to be a contraindication for lung transplant listing. The Oncologist 2017;22:620-622.

Visualizing the Propagation of Acute Lung Injury.

BACKGROUND: Mechanical ventilation worsens acute respiratory distress syndrome, but this secondary "ventilator-associated" injury is variable and difficult to predict. The authors aimed to visualize the propagation of such ventilator-induced injury, in the presence (and absence) of a primary underlying lung injury, and to determine the predictors of propagation. METHODS: Anesthetized rats (n = 20) received acid aspiration (hydrochloric acid) followed by ventilation with moderate tidal volume (V(T)). In animals surviving ventilation for at least 2 h, propagation of injury was quantified by using serial computed tomography. Baseline lung status was assessed by oxygenation, lung weight, and lung strain (V(T)/expiratory lung volume). Separate groups of rats without hydrochloric acid aspiration were ventilated with large (n = 10) or moderate (n = 6) V(T). RESULTS: In 15 rats surviving longer than 2 h, computed tomography opacities spread outward from the initial site of injury. Propagation was associated with higher baseline strain (propagation vs. no propagation [mean ± SD]: 1.52 ± 0.13 vs. 1.16 ± 0.20, P < 0.01) but similar oxygenation and lung weight. Propagation did not occur where baseline strain was less than 1.29. In healthy animals, large V(T) caused injury that was propagated inward from the lung periphery; in the absence of preexisting injury, propagation did not occur where strain was less than 2.0. CONCLUSIONS: Compared with healthy lungs, underlying injury causes propagation to occur at a lower strain threshold and it originates at the site of injury; this suggests that tissue around the primary lesion is more sensitive. Understanding how injury is propagated may ultimately facilitate a more individualized monitoring or management.

The Optical Biopsy: A Novel Technique for Rapid Intraoperative Diagnosis of Primary Pulmonary Adenocarcinomas.

BACKGROUND: With increasing use of chest computed tomography scans, indeterminate pulmonary nodules are frequently detected as an incidental finding and present a diagnostic challenge. Tissue biopsy followed by histological review and immunohistochemistry is the gold standard to obtain a diagnosis and the most common malignant finding is a primary lung adenocarcinoma. Our objective was to determine whether an intraoperative optical biopsy (molecular imaging) may provide an alternative approach for determining if a pulmonary nodule is a primary lung adenocarcinoma. METHODS: Before surgery, 30 patients with an indeterminate pulmonary nodule were intravenously administered a folate receptor-targeted fluorescent contrast agent specific for primary lung adenocarcinomas. During surgery, the nodule was removed and the presence of fluorescence (optical biopsy) was assessed in the operating room to determine if the nodule was a primary pulmonary adenocarcinoma. Standard-of-care frozen section and immunohistochemical staining on permanent sections were then performed as the gold standard to validate the results of the optical biopsy. RESULTS: Optical biopsies identified 19 of 19 (100%) primary pulmonary adenocarcinomas. There were no false positive or false negative diagnoses. An optical biopsy required 2.4 minutes compared to 26.5 minutes for frozen section (P < 0.001) and it proved more accurate than frozen section in diagnosing lung adenocarcinomas. CONCLUSIONS: An optical biopsy has excellent positive predictive value for intraoperative diagnosis of primary lung adenocarcinomas. With refinement, this technology may prove to be an important supplement to standard pathology for examining close surgical margins, identifying lymph node involvement, and determining whether suspicious nodules are malignant.

Molecular imaging to identify tumor recurrence following chemoradiation in a hostile surgical environment.

Surgical biopsy of potential tumor recurrence is a common challenge facing oncologists, surgeons, and cancer patients. Imaging modalities have limited ability to accurately detect recurrent cancer in fields affected by previous surgery, chemotherapy, or radiation. However, definitive tissue diagnosis is often needed to initiate treatment and to direct therapy. We sought to determine if a targeted fluorescent intraoperative molecular imaging technique could be applied in a clinical setting to assist a surgical biopsy in a "hostile" field. We describe the use of a folate-fluorescein conjugate to direct the biopsy of a suspected recurrent lung adenocarcinoma invading the mediastinum that had been previously treated with chemoradiation. We found that intraoperative imaging allowed the identification of small viable tumor deposits that were otherwise indistinguishable from scar and necrosis. Our operative observations were confirmed by histology, fluorescence microscopy, and immunohistochemistry. Our results demonstrate one possible application and clinical value of intraoperative molecular imaging.

Metabolic spectroscopy of inflammation in a bleomycin-induced lung injury model using hyperpolarized 1-(13) C pyruvate.

Metabolic activity in the lung is known to change in response to external insults, inflammation, and cancer. We report measurements of metabolism in the isolated, perfused rat lung of healthy controls and in diseased lungs undergoing acute inflammation using hyperpolarized 1-(13) C-labeled pyruvate. The overall apparent activity of lactate dehydrogenase is shown to increase significantly (on average by a factor of 3.3) at the 7 day acute stage and to revert substantially to baseline at 21 days, while other markers indicating monocarboxylate uptake and transamination rate are unchanged. Elevated lung lactate signal levels correlate well with phosphodiester levels as determined with (31) P spectroscopy and with the presence of neutrophils as determined by histology, consistent with a relationship between intracellular lactate pool labeling and the density and type of inflammatory cells present. We discuss several alternate hypotheses, and conclude that the most probable source of the observed signal increase is direct uptake and metabolism of pyruvate by inflammatory cells and primarily neutrophils. This signal is seen in high contrast to the low baseline activity of the lung.

Three-Dimensional Near-Infrared Specimen Mapping Can Identify the Distance from the Tumor to the Surgical Margin During Resection of Pulmonary Ground Glass Opacities.

BACKGROUND: Lung cancers can recur locally due to inadequate resection margins. Achieving adequate margin distances is challenging in pulmonary ground glass opacities (GGOs) because they are not easily palpable. To improve margin assessment during resection of GGOs, we propose a novel technique, three-dimensional near-infrared specimen mapping (3D-NSM). METHODS: Twenty patients with a cT1 GGO were enrolled and received a fluorescent tracer preoperatively. After resection, specimens underwent 3D-NSM in the operating room. Margins were graded as positive or negative based upon fluorescence at the staple line. Images were analyzed using ImageJ to quantify the distance from the tumor edge to the nearest staple line. This margin distance calculated by 3D-NSM was compared to the margin distance reported on final pathology several days postoperatively. RESULTS: 3D-NSM identified 20/20 GGOs with no false positive or false negative diagnoses. Mean fluorescence intensity for lesions was 110.92 arbitrary units (A.U.) (IQR: 77.77-122.03 A.U.) compared to 23.68 A.U. (IQR: 19.60-27.06 A.U.) for background lung parenchyma (p < 0.0001). There were 4 tumor-positive or close margins in the study cohort, and all 4 (100%) were identified by 3D-NSM. 3D-NSM margin distances were nearly identical to margin distances reported on final pathology (R2 = 0.9362). 3D-NSM slightly under-predicted margin distance, and the median difference in margins was 1.9 mm (IQR 0.5-4.3 mm). CONCLUSIONS: 3D-NSM rapidly localizes GGOs by fluorescence and detects tumor-positive or close surgical margins. 3D-NSM can accurately quantify the resection margin distance as compared to formal pathology, which allows surgeons to rapidly determine whether sublobar resection margin distances are adequate.

Anterior Mediastinal Neuroblastoma Associated with Syndrome of Inappropriate Antidiuretic Hormone Secretion: A Morphologic, Immunohistochemical, and Genetic Case Report and Review of the Literature.

We report a mediastinal neuroblastoma in an octogenarian with paraneoplastic syndrome of inappropriate antidiuretic hormone secretion (SIADH). Neuroblastomas are very rare tumors in adults, with thoracic or mediastinal locations being especially uncommon. These neoplasms have been occasionally associated with the SIADH. Given the rarity of incidence and paucity of diagnostic and outcomes data, the significance of standard neuroblastoma prognostic characteristics is unclear, and no treatment paradigms exist for these patients. Further studies are needed to inform future clinical guidelines.

Severe Acute Cellular Rejection With High-Grade Lymphocytic Bronchiolitis Following Transition from Tacrolimus to Belatacept in a Lung Transplantation Recipient: A Case Report.

This case report describes a lung transplantation recipient who developed severe acute cellular rejection with high-grade lymphocytic bronchiolitis after transition to a calcineurin-free regimen using belatacept. A 53-year-old man who had undergone lung transplantation 3 years prior developed progressive chronic kidney disease related to tacrolimus. He was transitioned off tacrolimus to belatacept to prevent the need for dialysis. He was admitted 2 months later with acute hypoxemic respiratory failure. Video-assisted thoracic surgery biopsy showed acute fibrinous and organizing pneumonia and A4B2 rejection. He subsequently developed chronic lung allograft dysfunction. This case illustrates the potential increased risk of acute rejection associated with belatacept maintenance immunosuppression.

Epithelial cell size dysregulation in human lung adenocarcinoma.

Human cells tightly control their dimensions, but in some cancers, normal cell size control is lost. In this study we measure cell volumes of epithelial cells from human lung adenocarcinoma progression in situ. By leveraging artificial intelligence (AI), we reconstruct tumor cell shapes in three dimensions (3D) and find airway type 2 cells display up to 10-fold increases in volume. Surprisingly, cell size increase is not caused by altered ploidy, and up to 80% of near-euploid tumor cells show abnormal sizes. Size dysregulation is not explained by cell swelling or senescence because cells maintain cytoplasmic density and proper organelle size scaling, but is correlated with changes in tissue organization and loss of a novel network of processes that appear to connect alveolar type 2 cells. To validate size dysregulation in near-euploid cells, we sorted cells from tumor single-cell suspensions on the basis of size. Our study provides data of unprecedented detail for cell volume dysregulation in a human cancer. Broadly, loss of size control may be a common feature of lung adenocarcinomas in humans and mice that is relevant to disease and identification of these cells provides a useful model for investigating cell size control and consequences of cell size dysregulation.

First-in-human results of targeted intraoperative molecular imaging for visualization of ground glass opacities during robotic pulmonary resection.

Background: Identifying ground glass opacities (GGOs) is challenging during robot-assisted thoracic surgery (RATS). Intraoperative molecular imaging (IMI) using tumor-targeted fluorescent tracers may address this clinical problem, but has never been evaluated in RATS. In a pilot study, we sought to determine whether IMI during RATS (RIMI) can localize GGOs. Methods: Ten patients with a cT1 GGO were enrolled. Prior to resection, participants received a folate-receptor targeted fluorescent tracer (OTL38). During RATS, a white-light robotic scope was utilized to identify tumors. RIMI was then conducted using a RATS thoracoscope with a wavelength-specific camera. Finally, a video-assisted thoracic surgery (VATS) thoracoscope designed to detect OTL38 was used as a control to compare to RIMI. The lesions were then resected under RIMI guidance. Results: By white-light robotic scope, 7/10 (70%) GGOs were visually identifiable by pleuroparenchymal distortions. RIMI identified tumor-specific fluorescence in all (100%) subjects. RIMI clearly located the three nodules that could not be seen by robotic white-light imaging. The mean fluorescence intensity (MFI) of tumors was 99.48 arbitrary units (A.U.) (IQR, 75.72-130.49 A.U.), which was significantly higher than background tissue with mean MFI 20.61 A.U. (IQR, 13.49-29.93 A.U., P<0.0001). Mean signal-to-background ratio was 5.71 (range, 2.28-10.13). When compared to VATS-IMI as a control, there were no significant differences in MFI of tumors, background tissue, or signal-to-background ratios. In summary, RIMI compared favorably to VATS-IMI by all measured imaging characteristics. Conclusions: RIMI is feasible for identification of GGOs during robotic resection as compared to white light thoracoscopy and compares favorably to VATS-IMI.

Tracheobronchopathia osteochondroplastica presenting as a single dominant tracheal mass.

Tracheobronchopathia osteochondroplastica is a rare, benign disorder of upper airways characterized by multiple submucosal metaplastic cartilaginous and bony nodules arising from the tracheal cartilage. We report an unusual presentation of tracheobronchopathia osteochondroplastica as a single dominant nodule arising from the anterior tracheal rings in a young adult man who presented with wheezing and symptoms of airway obstruction. The differential diagnosis of cartilaginous and bony endotracheal lesions is discussed.

Molecular imaging can identify the location to perform a frozen biopsy during intraoperative frozen section consultation.

BACKGROUND: Intraoperative frozen section (FS) consultation is an important tool in surgical oncology that suffers from sampling error because the pathologist does not always know where to perform a biopsy of the surgical specimen. Intraoperative molecular imaging is a technology used in the OR to visualize lesions during surgery. We hypothesized that molecular imaging can address this pathology challenge in FS by visualizing the cancer cells in the specimen in the pathology suite. Here, we report the development and validation of a molecular-imaging capable cryostat called Smart-Cut. METHODS: A molecular imaging capable cryostat prototype was developed and tested using a murine model. Tumors grown in mice were targeted with a NIR contrast agent, indocyanine green (ICG), via tail vein injection. Tumors and adjacent normal tissue samples were frozen sectioned with Smart-Cut. Fluorescent sections and non-fluorescent sections were prepared for H&E and fluorescent microscopy. Fluorescent signal was quantified by tumor-to-background ratio (TBR). NIR fluorescence was tested in one patient enrolled in a clinical trial. RESULTS: The Smart-Cut prototype has a small footprint and fits well in the pathology suite. Fluorescence imaging with Smart-Cut identified cancerous tissue in the specimen in all 12 mice. No false positives or false negatives were seen, as confirmed by H&E. The mean TBR in Smart-Cut positive tissue sections was 6.8 (SD±3.8). In a clinical application in the pathology suite, NIR imaging identified two lesions in a pulmonary resection specimen, where traditional grossing only identified one. CONCLUSION: Molecular imaging can be integrated into the pathology suite via the Smart-Cut device, and can detect cancer in frozen tissue sections using molecular imaging in a murine model.

Analysis of Epstein-Barr virus glycoprotein B functional domains via linker insertion mutagenesis.

Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.