Vascular invasion identifies the most aggressive histologic subset of stage I lung adenocarcinoma: Implications for adjuvant therapy.

OBJECTIVES: Approximately 15% of stage I lung adenocarcinomas will recur despite adequate surgical therapy. Adjuvant therapy may benefit specific high-risk subsets; however, it is unclear which patients are sufficiently predisposed to recurrence to warrant intensified therapy. MATERIALS AND METHODS: 517 AJCC 8th edition stage I/0 lung adenocarcinomas ≤ 4 cm total size were graded (WHO-2015 and WHO-2021) and compared to stage subgroupings using 7-year recurrence free (RFS), disease specific (DSS), and overall survival (OS). Low malignant potential (LMP) adenocarcinoma was assigned as previously defined. Univariate/multivariate analysis was performed to assess risk factors associated with aggressive behavior. RESULTS: Vascular invasion was the most significant histologic feature on multivariate analysis for both RFS (HR = 4.68, p < 0.001) and DSS (HR = 3.67, p = 0.001) and nearly reached significance for OS (HR = 1.47, p = 0.060). Angioinvasive adenocarcinomas comprised 26 % of the cohort and exhibited a 7-year 64 % RFS, 73 % DSS, and 50 % OS; in contrast to 20 % WHO-2015-G3 (7-year 71 % RFS, 79 % DSS, & 54 % OS), 44 % WHO-2021-G3 (7-year 79 % RFS, 85 % DSS, & 56 % OS), and 21 % stage IB (7-year 72 % RFS, 79 % DSS, and 50 % OS) adenocarcinomas. The majority (>50 %) of overall mortality was disease specific for angioinvasive adenocarcinoma whereas ≤25 % of overall mortality was disease specific for the remaining tumors. Angioinvasive adenocarcinomas were proportionally more common among those still smoking at diagnosis (49 %), male sex (49 %), and black race (16 %) than other subtypes. CONCLUSION: Patients with AJCC 8th ed. stage I angioinvasive lung adenocarcinomas are at high-risk of cancer-specific mortality and should be considered for clinical trials evaluating benefit of adjuvant therapy.

Surgeon Experience Is Associated With Prolonged Air Leak After Robotic-assisted Pulmonary Lobectomy.

BACKGROUND: Prolonged air leak (PAL) (>5 days) after robotic-assisted pulmonary lobectomy is a significant complication. This study aimed to determine patient- and surgeon-related factors that can predict PAL after robotic lobectomy for lung cancer. METHODS: This study was a retrospective review of a single-center experience of robotic-assisted lobectomy for lung cancer. Perioperative variables, including surgeon case experience, patient demographics, diffusion capacity of lung for carbon monoxide, forced expiratory volume in 1 second, body mass index, and smoking status were evaluated. RESULTS: A total of 305 robotic-assisted lobectomies performed by 4 surgeons met inclusion criteria from June 2016 to February 2019. The 30-day postoperative mortality was 1.2%. PAL developed in 27 of 305 (8.8%) patients. Surgeons' robotic experience was grouped by 10-case increments. When adjusted for age and sex, the odds for PAL decreased by 15% for every 10 robotic lobectomies the surgeons performed (odds ratio [OR], 0.85; 95% CI, 0.74-0.99; P = .0384). Logistic regression models showed a linear transition curve at the 50th case. Female sex (OR, 2.62; 95% CI, 1.03-6.69; P = .0314) and younger age (OR, 0.61; 95% CI, 0.41-0.91; P = .0184) were statistically significant risk factors for PAL. Cumulative sum analysis similarly showed a strong association between experience and PAL. Preoperative diffusing capacity of lung for carbon monoxide, forced expiratory volume in 1 second, body mass index, and smoking status were not statistically significant predictive factors. CONCLUSIONS: These results show that surgeon robotic case experience is associated with the rate of postoperative PAL: as the number of robotic lobectomies increases, the rate of PAL significantly decreases. It is imperative to emphasize that a learning curve exists for this approach that directly affects patient outcomes.

Conversion to Thoracotomy During Thoracoscopic vs Robotic Lobectomy: Predictors and Outcomes.

BACKGROUND: Conversion to thoracotomy during minimally invasive lobectomy for lung cancer is occasionally necessary. Differences between video-assisted thoracoscopic surgery (VATS) and robotic-assisted thoracoscopic surgery (RATS) lobectomy conversion have not been described. METHODS: We queried The Society of Thoracic Surgeons General Thoracic Surgery Database from January 1, 2015 to December 31, 2018. Patients with prior thoracic operations and metastatic disease were excluded. Univariable comparisons with χ2 and Kruskal-Wallis tests and multivariable logistic regression modeling were performed. RESULTS: There were 27,695 minimally invasive lobectomies from 269 centers. Conversion to thoracotomy occurred in 11.0% of VATS and 6.0% of RATS (P < .001). Conversion was associated with increased mortality (P < .001), major complications (P < .001), and intraoperative (P < .001) and postoperative (P < .001) blood transfusions. Conversion from RATS occurred emergently (P < .001) and for vascular injury (P < .001) more frequently than from VATS, but there was no difference in overall major complications or mortality. Mortality after conversion was 3.1% for RATS and 2.2% for VATS (P = .24). Clinical cancer stage II or III (P < .001), preoperative chemotherapy (P = .003), forced expiratory volume in 1 second (P = .006), body mass index (P < .001), and left-sided resection (P = .0002) independently predicted VATS conversion. For RATS clinical stage III (P = .037), left-sided resection (P = .041), and forced expiratory volume in 1 second (P = .002) predicted conversion. Lower volume centers had increased rates of conversion (P < .001) in both groups. CONCLUSIONS: Conversion from minimally invasive to open lobectomy is associated with increased morbidity and mortality. Conversion occurs more frequently during VATS compared with RATS, albeit less often emergently, and with similar rates of overall mortality and major complications. Predictors, urgency, and reasons for conversion differ between RATS and VATS lobectomy and may assist in patient selection.

Robotic-Assisted Complex Pulmonary Resection: Sleeve Lobectomy for Cancer.

The use of robotic assistance for complex pulmonary resections such as segmentectomy and sleeve lobectomy has steadily increased in recent years. These operations are technically challenging as they require fine dissection and suturing, which is often difficult to perform using traditional minimally invasive techniques. Robotic surgery is well-suited for complex pulmonary surgery given its specific advantages related to superior optics and precise tissue manipulation and dissection. Herein we describe our technique for robotic-assisted complex pulmonary surgery with a specific focus on right upper sleeve lobectomy for cancer, including associated video case demonstration. The principles discussed are generalizable to other complex lung and tracheobronchial operations and highlight the benefits of the robotic platform.

Surgery and invasive diagnostic procedures for benign disease are rare in a large low-dose computed tomography lung cancer screening program.

OBJECTIVE: Lung cancer screening with low-dose chest computed tomography improves survival. However, concerns about overdiagnosis and unnecessary interventions persist. We reviewed our lung cancer screening program to determine the rate of surgery and invasive procedures for nonmalignant disease. METHODS: We reviewed all patients undergoing lung cancer screening from January 2012 to June 2017 with follow-up through January 2019. Patients with suspicious findings (Lung CT Screening Reporting and Data System 4) were referred for further evaluation. RESULTS: Of 3280 patients screened, 345 (10.5%) had Lung CT Screening Reporting and Data System 4 findings. A total of 311 patients had complete follow-up, of whom 93 (29.9%) were diagnosed with lung cancer. Eighty-three patients underwent lung surgery (2.5% of screened patients). Forty patients underwent lobectomy (48.2%), 3 patients (3.6%) underwent bilobectomy, and 40 patients (48.2%) underwent sublobar resection. Fourteen patients underwent surgery for benign disease (0.43% of screened patients). Fifty-four patients, 5 with benign disease, had at least 1 invasive diagnostic procedure but never underwent surgery. The incidence of any invasive intervention for nonmalignant disease was 0.95% (31/3280 patients). There were no postprocedural deaths within 60 days. Twenty-five patients (0.76%) underwent stereotactic body radiation therapy; 19 patients (76%) had presumed lung cancer without pretreatment pathologic confirmation. CONCLUSIONS: Surgical resection for benign disease occurred in 0.43% of patients undergoing lung cancer screening. The combined incidence of any invasive diagnostic or therapeutic intervention, including surgical resection, for benign disease was only 0.95%. Periprocedural complications were rare. These results indicate that concern over unnecessary interventions is overstated and should not hinder adoption of lung cancer screening. A multidisciplinary team approach, including thoracic surgeons, is critical to maintain an appropriate rate of interventions in lung cancer screening.

Pulmonary Adenocarcinomas of Low Malignant Potential: Proposed Criteria to Expand the Spectrum Beyond Adenocarcinoma In Situ and Minimally Invasive Adenocarcinoma.

Lung cancer screening has improved mortality among high-risk smokers but has coincidentally detected a fraction of nonprogressive adenocarcinoma historically classified as bronchoalveolar carcinoma (BAC). In the National Lung Screening Trial (NLST) the majority of BAC-comprising 29% of computed tomography-detected stage I lung adenocarcinoma-were considered overdiagnosis after extended follow-up comparison with the control arm. In the current classification, adenocarcinoma in situ and minimally invasive adenocarcinoma have replaced BAC but together comprise only ∼5% of stage I lung adenocarcinoma. Lepidic and subsets of papillary and acinar adenocarcinoma also infrequently recur. We, therefore, propose criteria for low malignant potential (LMP) adenocarcinoma among nonmucinous adenocarcinoma measuring ≤3 cm in total, exhibiting ≥15% lepidic growth, and lacking nonpredominant high-grade patterns (≥10% cribriform, ≥5% micropapillary, ≥5% solid), >1 mitosis per 2 mm2, angiolymphatic or visceral pleural invasion, spread through air spaces or necrosis. We tested these criteria in a multi-institutional cohort of 328 invasive stage I (eighth edition) and in situ adenocarcinomas and observed 16% LMP and 7% adenocarcinoma in situ/minimally invasive adenocarcinoma which together (23%) approximated the frequency of overdiagnosed stage I BAC in the NLST. The LMP group had 100% disease-specific survival. The proposed LMP criteria, incorporating multiple histologic parameters, may be a clinically useful "low-grade" prognostic group. Validation of these criteria in additional retrospective cohorts and prospective screen-detected cohorts should be considered.

Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome.

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Antiadrenergic autoimmunity in postural tachycardia syndrome.

AIMS: Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and ß1/2-adrenergic receptors (ß1/2AR). METHODS AND RESULTS: Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and ß1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and ß1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, ß1AR and ß2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and ß1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the ß1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated ß1AR and α1AR activity but not with ß2AR activity. CONCLUSION: These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and ß1AR responsiveness are important in the pathophysiology of postural tachycardia.

Significantly reduced lymphadenopathy, salivary gland infiltrates and proteinuria in MRL-lpr/lpr mice treated with ultrasoluble curcumin/turmeric: increased survival with curcumin treatment.

OBJECTIVES: Commercial curcumin (CU), derived from food spice turmeric (TU), has been widely studied as a potential therapeutic for a variety of oncological and inflammatory conditions. Lack of solubility/bioavailability has hindered curcumin's therapeutic efficacy in human diseases. We have solubilised curcumin in water applying heat/pressure, obtaining up to 35-fold increase in solubility (ultrasoluble curcumin (UsC)). We hypothesised that UsC or ultrasoluble turmeric (UsT) will ameliorate systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS)-like disease in MRL-lpr/lpr mice. METHODS: Eighteen female MRL-lpr/lpr (6 weeks old) and 18 female MRL-MpJ mice (6 weeks old) were used. Female MRL-lpr/lpr mice develop lupus-like disease at the 10th week and die at an average age of 17 weeks. MRL-MpJ mice develop lupus-like disease around 47 weeks and typically die at 73 weeks. Six mice of each strain received autoclaved water only (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group) or UsT (lpr-TU or MpJ-TU group) in the water bottle. RESULTS: UsC or UsT ameliorates SLE in the MRL-lpr/lpr mice by significantly reducing lymphoproliferation, proteinuria, lesions (tail) and autoantibodies. lpr-CU group had a 20% survival advantage over lpr-water group. However, lpr-TU group lived an average of 16 days shorter than lpr-water group due to complications unrelated to lupus-like illness. CU/TU treatment inhibited lymphadenopathy significantly compared with lpr-water group (p=0.03 and p=0.02, respectively) by induction of apoptosis. Average lymph node weights were 2606±1147, 742±331 and 385±68 mg, respectively, for lpr-water, lpr-CU and lpr-TU mice. Transferase dUTP nick end labelling assay showed that lymphocytes in lymph nodes of lpr-CU and lpr-TU mice underwent apoptosis. Significantly reduced cellular infiltration of the salivary glands in the lpr-TU group compared with the lpr-water group, and a trend towards reduced kidney damage was observed in the lpr-CU and lpr-TU groups. CONCLUSIONS: These studies show that UsC/UsT could prove useful as a therapeutic intervention in SLE/SS.

Parafilm-M®, An Available Cost-Effective Alternative for Immuno-blot Pouches.

Commercially available standard immuno-blot pouches do play an efficient role in antibody incubation in performing an immuno-blot, but are not readily available in the laboratory and have to be specifically ordered. We have developed an equally efficient technique to make an immune-blot more cost-effective with more conservation of antibodies by using a common and readily available laboratory product Parafilm-M(®). Parafilm-M(®) which serves as a sealant for various items of laboratory equipment can be used for antibody incubation. Manually made Parafilm-M(®) pouch has a clear advantage over standard immuno-blot pouches in terms of availability, cost-effectiveness, and consumption of antibodies that ultimately reduces the cost of an immuno-blot. We have performed a series of experiments to check the efficacy of both the techniques. Samples with equal amount of protein were analyzed on separate SDS PAGE gels. The proteins were transferred electrophoretically to the nitrocellulose membrane using Trans-Blot(®) Turbo™ Mini Nitrocellulose Transfer Pack. Antibody incubation was done using standard immuno-blot pouch, standard container and Parafilm-M(®) sealed pouch. The expression of protein was determined and the results of immuno-blots were compared. We found that antibodies are binding the membrane in Parafilm-M(®) pouches as efficiently as in container method or in standard immuno-blot pouches. By restricting the membrane, the surface area of the manually made Parafilm-M(®) pouch can be reduced, less diluent is required to cover the membrane as a result less antibodies are consumed. We also calculated that each immuno-blot pouch cost around $0.1906, whereas the cost for Parafilm-M(®) pouch is 0.0695 which is almost one-third the price of an immuno-blot pouch. Thus, Parafilm-M(®) method distinctly provides a cost-effective solution for antibody incubation.

Single-cell western blotting.

Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

Independent ventilation of the graft and native lungs in vivo after rat lung transplantation.

Rat lung transplantation is a proven experimental technique for the study of lung injury following lung transplantation. We have modified the surgical and ventilatory techniques to allow for independent ventilation in vivo of the transplanted graft and native lungs. This will provide additional data on the physiology and function of the transplanted graft and ameliorate the problem of progressive graft lung collapse and thereby allow for an improved model of ischemia-reperfusion injury and ventilator-induced lung injury in the setting of lung transplantation.

Caspase inhibition improves ischemia-reperfusion injury after lung transplantation.

Ischemia-reperfusion injury is associated with cell death in many organ systems. The role of programmed cell death (PCD) pathways and the ultimate clinical relevance of PCD in the context of lung transplantation (LTx) are unknown. In randomized and blinded studies, rat single LTx was performed in the presence of caspase inhibitors after 'short' (6 h) and 'long' (18 h) periods of cold ischemic storage. Lung function, electron microscopic morphology, caspase 3, 8 and 9 activities and TUNEL assays were evaluated. Endothelial cells and lymphocytes were observed undergoing apoptotic cell death with electron microscopy. Caspase activities were significantly up-regulated immediately after the initial flush and increased further during short periods of cold ischemic storage. A significant amount of apoptotic cell death was observed after LTx and reperfusion. Caspase inhibition virtually eliminated apoptotic cell death and led to improved lung function after LTx and reperfusion. Activation of caspases during cold ischemia contributes significantly to cell death in LTx. Suppression of caspase activity appears to decrease apoptosis and improve lung function. Clearly, this needs to be investigated further with more experiments to validate the potential role of caspase inhibition as a therapeutic modality in ischemia-reperfusion-induced lung injury.

Intralesional radioimmunotherapy with Yttrium-90-labeled human monoclonal IgM in nude mice bearing human tumor xenografts.

UNLABELLED: Biodistribution studies demonstrate that intralesional administration of radiolabeled IgM results in high retention of radioactivity with little normal tissue uptake. This study examines the therapeutic potential of this modality. MATERIALS AND METHODS: Nude mice bearing subcutaneous human head and neck squamous cell carcinoma xenografts were treated with single intralesional (IL) injections of tumor-reactive human monoclonal IgM (CR4E8) labeled with 25-394 microCi of yttrium-90 (90Y). Untreated mice, mice treated with IL unlabeled immunoconjugate or IL 90Y-aggregate, 160-400 microCi, served as controls. Mice were monitored for tumor growth and toxicity. RESULTS: Mice received 80 Gy per 100 microCi of 90Y-labeled IgM and 110 Gy per 100 microCi of 90Y-aggregate. All tumors treated with 90Y-labeled IgM responded. In mice that received > or = 100 microCi, tumors macroscopically disappeared within three weeks from treatment with seventy-six percent tumor-free at 216 days. Acute toxicities associated with high activity 90Y-labeled IgM and 90Y-aggregate were moist skin desquamation and reduced blood counts. Late radiation damage to connective tissue was observed in mice treated with > 100 microCi of 90Y-labeled IgM. CONCLUSIONS: Intralesional administration of 90Y-labeled IgM can ablate tumors in nude mice with modest acute or late toxicity. Further development of this modality for loco-regional therapy is warranted.

QT dispersion and early arrhythmic risk in acute myocardial infarction.

BACKGROUND: This study sought to find out QT dispersion in healthy individuals and patients of acute myocardial infarction and to find correlation, if any, between QT dispersion and the incidence of ventricular arrhythmias in acute myocardial infarction. METHODS AND RESULTS: QT dispersion was calculated from a 12-lead electrocardiogram in 100 patients of acute myocardial infarction admitted in intensive coronary care unit and 100 age- and sex-matched healthy individuals. In patients of acute myocardial infarction, QT dispersion was calculated on admission, 24 hours after admission and at the time of discharge from intensive coronary care unit. Average QT dispersion in acute myocardial infarction was found to be significantly higher on admission (76.4 +/- 18.3 ms), 24 hours after admission (62.88 +/- 17.52 ms) and at the time of discharge from intensive coronary care unit (51.79 +/- 16.79 ms) than in healthy individuals (29.76 +/- 6.06 ms; p<0.05). QT dispersion was found to be significantly increased in patients of acute myocardial infarction with ventricular arrhythmias (82.06 +/- 16.86 ms) than in those without (66.75 +/- 16.28 ms; p<0.01). Patients of acute myocardial infarction with ventricular tachycardia or ventricular fibrillation had significantly increased QT dispersion (96.25 +/- 15.97 ms) than those who had only ventricular premature beats (80 +/- 15.04 ms; p<0.01). QT dispersion was found to be significantly greater in patients with anterior wall acute myocardial infarction (79.80 +/- 18.19 ms) than in those with inferior wall acute myocardial infarction (71.9 +/- 17.48 ms; p<0.05). At the time of discharge from intensive coronary care unit no statistically significant difference was found in QT dispersion in those who received thrombolysis (51.58 +/- 16.05 ms) and those who did not (48.18 +/- 14.68 ms; p>0.05). QT dispersion was found to be significantly higher in those who died (88.66 +/- 15.97 ms) than in those who survived (74.23 +/- 17.91 ms; p<0.05). QT dispersion was significantly higher in ventricular arrhythmic deaths (97.14 +/- 17.04 ms) than those who had non-arrhythmiac deaths (81.25 +/- 11.25 ms; p<0.05). CONCLUSIONS: Interlead QT variation and its measure as QT dispersion challenges our current approach to the electrocardiographic assessment of arrhythmic risk. QT dispersion may provide a potentially simple, cheap, non-invasive method of measuring underlying dispersion of ventricular excitability.