CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

Cancer stem cells (CSCs) are a subpopulation with the properties of extensive self-renewal, capability to generate differentiated cancer cells and resistance to therapies. We have previously shown that malignant pleural effusions (MPEs) from patients with non-small-cell lung cancer (NSCLC) represent a valuable source of cancer cells that can be grown as three-dimensional (3D) spheroids enriched for stem-like features, which depend on the activation of the Yes-associated protein-transcriptional coactivator with PDZ-binding motif (YAP-TAZ)/Wnt-ßcatenin/stearoyl-CoA desaturase 1 (SCD1) axis. Here, we describe a novel support, called CytoMatrix, for the characterization of limited amounts of cancer cells isolated from MPEs of patients with NSCLC. Our results show that this synthetic matrix allows an easy and fast characterization of several epithelial cellular markers. The use of CytoMatrix to study CSCs subpopulation confirms that SCD1 protein expression is enhanced in 3D spheroids when compared with 2D adherent cell cultures. YAP/TAZ nuclear-cytoplasmic distribution analysed by CytoMatrix in 3D spheroids is highly heterogeneous and faithfully reproduces what is observed in tumour biopsies. Our results confirm and extend the robustness of our workflow for the isolation and phenotypic characterization of primary cancer cells derived from the lung MPEs and underscore the role of SCD1.

Prognostic Impact of Node-Spreading Pattern in Surgically Treated Small-Cell Lung Cancer: A Multicentric Analysis.

OBJECTIVE: Although surgery in selected small-cell lung cancer (SCLC) patients has been proposed as a part of multimodality therapy, so far, the prognostic impact of node-spreading pattern has not been fully elucidated. To investigate this issue, a retrospective analysis was performed. METHODS: From 01/1996 to 12/2012, clinico-pathological, surgical, and oncological features were retrospectively reviewed in a multicentric cohort of 154 surgically treated SCLC patients. A multivariate Cox proportional hazard model was developed using stepwise regression, in order to identify independent outcome predictors. Overall (OS), cancer-specific (CSS), and Relapse-free survival (RFS) were calculated by Kaplan-Meier method. RESULTS: Overall, median OS, CSS, and RFS were 29 (95 % CI 18-39), 48 (95 % CI 19-78), and 22 (95 % CI 17-27) months, respectively. Lymphadenectomy was performed in 140 (90.9 %) patients (median number of harvested nodes: 11.5). Sixty-seven (47.9 %) pN0-cases experienced the best long-term survival (CSS: 71, RFS: 62 months; p < 0.0001). Among node-positive patients, no prognostic differences were found between pN1 and pN2 involvement (CSS: 22 vs. 15, and RFS: 14 vs. 10 months, respectively; p = 0.99). By splitting node-positive SCLC according to concurrent N1-invasion, N0N2-patients showed a worse CSS compared to those cases with combined N1N2-involvement (N0N2: 8 months vs. N1N2: 22 months; p = 0.04). On the other hand, the number of metastatic stations (p = 0.80) and the specific node-level (p = 0.85) did not affect CSS. At multivariate analysis, pN+ (HR: 3.05, 95 % CI 1.21-7.67, p = 0.02) and ratio between metastatic and resected lymph-nodes (RL, HR: 1.02, 95 % CI 1.00-1.04, p = 0.03) were independent predictors of CSS. Moreover, node-positive patients (HR: 3.60, 95 % CI 1.95-6.63, p < 0.0001) with tumor size ≥5 cm (HR: 1.85, 95 % CI 0.88-3.88, p = 0.10) experienced a worse RFS. CONCLUSIONS: In selected surgically treated SCLC, the long-term survival may be stratified according to the node-spreading pattern.

Induction Therapy versus Initial Surgery in Advanced Thymic Tumors: Perioperative and Oncological Outcome.

Background Despite the intense debate concerning management of advanced thymic tumors, no specific oncological strategies have been yet recommended. We report our 13 years' experience to investigate this issue. Methods From 01/2001 to 12/2013, the clinical data of 28 patients treated for Masaoka stages III-IV thymic tumors were retrospectively reviewed. Eleven potentially nonresectable patients (Group A) underwent induction chemotherapy plus surgery, while immediate surgery was performed in 17 patients (Group B). The endpoint was to compare the two groups on (1) surgical resectability; (2) postoperative course; (3) disease-free survival; and (4) overall survival. Results Both groups were comparable in terms of age, gender, clinical stage, clinical tumor size, histology, and adjuvant therapy. Length of surgery was statistically longer in Group A (p = 0.015). Combined surgery and R0 resection was similarly performed in both groups (p = 0.14 and p = 0.99, respectively). The 3-year overall survival was 71.4% for Group A and 93.3% for Group B (p = 0.84). On the other hand, 3-year disease-free survival was 40.5 and 53.7% for Group A and B, respectively (p = 0.67). At multivariate analysis, gender was the strongest predictor for recurrence (hazard ratio = 5.71 [1.22; 26.67], p = 0.03). Conclusion Our results suggest that induction therapy allows obtaining acceptable clinical responses as well as resectability, survival, and recurrence rates. In selected patients with "clinically resectable" stage III-IV cancers, surgery (as first step of a multimodality therapy) could be a feasible treatment option.

Prognostic role of serum p53 antibodies in lung cancer.

BACKGROUND: Mutations in the TP53 (Tumour Protein 53) gene can lead to expression of mutant p53 proteins that accumulate in cancer cells and can induce circulating p53 antibodies in cancer patients. Our aim was to evaluate the presence and prognostic role of these antibodies in lung cancer patients and to investigate whether they were related to p53 expression or TP53 mutations in tumour tissues. METHODS: A total of 201 lung cancer patients were evaluated for p53 antibodies by ELISA (Enzyme-Linked Immunosorbent Assay) and control was obtained from 54 patients with non-malignant disorders; p53 expression was evaluated in 131 of the lung cancer patients by immunohistochemistry and TP53 mutations were then investigated in 53 tumours positively staining for p53 and in 12 tumours without p53 overexpression, whose DNA was available for direct sequencing. RESULTS: Our results show that 20.4% of cancer patients have positive levels of p53 antibodies, while none of the controls resulted positive. High levels of p53 expression are detected in 57.3% of cases and a significant correlation between serum p53 antibodies and high levels of p53 expression in the corresponding tumours is observed. In non-small cell lung cancer, p53 antibodies are significantly associated with poorly differentiated tumours; furthermore, high levels of p53 expression significantly correlated with squamous cell carcinoma and tumours with highest grade. Survival time of non-small cell lung cancer patients low/negative for serum p53 antibodies was significantly longer compared to patients with positive levels (p = 0.049); in particular, patients with squamous cell carcinoma, but not adenocarcinoma, low/negative for these antibodies show a significant better survival compared to serum-positive patients (p = 0.044). CONCLUSIONS: In our study, detection of serum p53 antibodies in non-small cell lung cancer patients has been shown to be useful in identifying subsets of patients with poor prognosis. A significant correlation between the presence of serum p53 antibodies in lung cancer patients and p53 overexpression in the corresponding tumours was also observed. We did not find a significant correlation between levels of serum p53 antibodies and TP53 mutations in the corresponding tumours.

Multimodal evaluation of locoregional anaesthesia efficacy on postoperative pain after robotic pulmonary lobectomy for NSCLC: a pilot study.

The primary objectives of the study were to analyse the robotic approach and ultrasound-guided paravertebral block compared to thoracoscopic intercostal nerve block after robotic pulmonary lobectomy on postoperative pain and opioids use. The secondary objectives were to analyse and compare patients' necessity of additional antalgic drugs and patients' performance during respiratory therapy, following robotic surgery and in the two groups. Consecutively, 52 patients undergoing robotic pulmonary lobectomies were treated either with ropivacaine-based intercostal nerve block or paravertebral block from February 2022 to October 2022. When necessary, morphine was administered at day 1. Acetaminophen was administered as an additional antalgic drug on demand up to 3 g per day. Pain was measured 1 h after the end of the surgical procedure and daily through the pain numeric rating scale (NRS). Morphine administration rate and per day and total additional administrations of acetaminophen were recorded. Pain and opioids administration was measured 1 month after the procedure. Data were analysed in the overall population and in the intercostal nerve block group VS paravertebral block group. Overall, 34.6% of the patients required morphine administration and 51.7% of the patients required at least daily acetaminophen administration up to discharge. At 1 month postoperatively, four patients presented with chronic pain and one still was under opioid medication. At intergroup analysis, the paravertebral block group demonstrated lower NRS at fixed time points (p < 0.0001) and lower morphine consumption (45.7%VS11.8%; p = 0.02). Acetaminophen rescue administration at fixed time points was lower in the paravertebral block group (p < 0.0001) and mobility and dynamic pain resulted in better results (p = 0.03; p = 0.04). At 1 month, no differences were found between study groups. Similarly to other minimally invasive techniques, postoperative pain may arise after robotic pulmonary lobectomy. Paravertebral bloc can help to reduce postoperative pain as well as morphine and antalgic drugs administration and improve early mobilization.

Analysis of predictive factors of unforeseen nodal metastases in resected clinical stage I NSCLC.

Background: Despite notable advances made in preoperative staging, unexpected nodal metastases after surgery are still significantly detected. In this study we aim to analyze the upstaging rate in patients with clinical stage I NSCLC without evidence of nodal disease in the preoperative staging who underwent lobectomy and radical lymphadenectomy. Methods: Patients who underwent lobectomy and systematic lymphadenectomy for clinical stage I NSCLC were evaluated. Exclusion criteria included the neoadjuvant treatment, incomplete resection and no adherence to preoperative guidelines. Results: A total of 297 patients were included in the study. 159 patients were female, and the median age was 68 (61 - 73). The variables that showed a significant correlation with the upstaging rate at the univariate analysis were the number of resected lymph nodes and micropapillar/solid adenocar-cinoma subtype. This result was confirmed in the multivariate analysis with a OR= 2.545 (95%CI 1.136-5.701; p=0.02) for the number of resected lymph nodes and a OR=2.717 (95%CI 1.256-5.875; p=0.01) for the high-grade pattern of adenocarcinoma. Conclusion: Our results showed that in a homogeneous cohort of patients with clinical stage I NSCLC, the number of resected lymph nodes and the histological subtype of adenocarcinoma can significantly be associated with nodal metastasis.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Uniportal thoracoscopic left main bronchus resection with new lobar carina reconstruction.

Isolated endobronchial tumors of the left main bronchus are quite rare. They are treated by surgical resection, preferably using lung-sparing techniques, when possible. These procedures are technically challenging and are usually performed through a standard thoracotomy.  This video tutorial shows a left main bronchus sleeve resection with distal lobar carina reconstruction, performed through a uniportal thoracoscopic approach, for a typical carcinoid tumor of the distal left main bronchus.

Thymic Epithelial Tumors as a Model of Networking: Development of a Synergistic Strategy for Clinical and Translational Research Purposes.

Among the group of thymic epithelial tumors (TET), thymomas often show either uncertain or explicit malignant biological behavior, local invasiveness, and intrathoracic relapse and are often difficult to manage. From the initial stages, thymic carcinomas tend to show aggressive behavior and extrathoracic spread. Moreover, the interplay of epithelial cells and thymocytes in thymomas causes complex immune derangement and related systemic autoimmune diseases. Due to their rare occurrence and to the limited funding opportunities available for rare tumors, it is challenging to make advances in clinical and translational research in TET. The authors of this paper are all members of a multidisciplinary clinical and research thoracic tumor team. Strong input was given to the team by long-standing expertise in TET in the Pathology Department. In addition, thanks to the collaboration between research units at our Institute as well as to national collaborations, over the last 10 years we were able to perform several tissue-based research studies. The most recent studies focused on microRNA and on functional studies on the thymic carcinoma cell line 1889c. The recent implementation of our biobank now provides us with a new tool for networking collaborative research activities. Moreover, the participation in a worldwide community such as ITMIG (International Thymic Malignancy Interest Group) has allowed us to significantly contribute toward fundamental projects/research both in tissue-based studies (The Cancer Genome Atlas) and in clinical studies (TNM staging of TET). Our achievements derive from constant commitment and long-standing experience in diagnosis and research in TET. New perspectives opened up due to the establishment of national [the Italian Collaborative Group for ThYmic MalignanciEs (TYME)] and European reference networks such as EURACAN, for an empowered joint clinical action in adult solid rare tumors. The challenge we face still lies in the advancement of clinical and basic science in thymic epithelial malignancies.

LINC00174 is a novel prognostic factor in thymic epithelial tumors involved in cell migration and lipid metabolism.

Long non-coding RNAs are emerging as new molecular players involved in many biological processes, such as proliferation, apoptosis, cell cycle, migration, and differentiation. Their aberrant expression has been reported in variety of diseases. The aim of this study is the identification and functional characterization of clinically relevant lncRNAs responsible for the inhibition of miR-145-5p, a key tumor suppressor in thymic epithelial tumors (TETs). Starting from gene expression analysis by microarray in a cohort of fresh frozen thymic tumors and normal tissues, we identified LINC00174 as upregulated in TET. Interestingly, LINC00174 expression is positively correlated with a 5-genes signature in TETs. Survival analyses, performed on the TCGA dataset, showed that LINC00174 and its associated 5-genes signature are prognostic in TETs. Specifically, we show that LINC00174 favors the expression of SYBU, FEM1B, and SCD5 genes by sponging miR-145-5p, a well-known tumor suppressor microRNA downregulated in a variety of tumors, included TETs. Functionally, LINC00174 impacts on cell migration and lipid metabolism. Specifically, SCD5, one of the LINC00174-associated genes, is implicated in the control of lipid metabolism and promotes thymic cancer cells migration. Our study highlights that LINC00174 and its associated gene signature are relevant prognostic indicators in TETs. Of note, we here show that a key controller of lipid metabolism, SCD5, augments the migration ability of TET cells, creating a link between lipids and motility, and highlighting these pathways as relevant targets for the development of novel therapeutic approaches for TET.

Intracellular presence of insulin and its phosphorylated receptor in non-small cell lung cancer.

Insulin has been known for a long time to influence the growth and differentiation of normal and transformed cells. In order to delineate the role of insulin specifically in non-small cell lung cancer (NSCLC), we have now searched by immunohistochemistry (IHC) for the presence of insulin in NSCLC samples. Among the 112 samples we studied, 30 were found to contain insulin, which was detected in the form of intracytoplasmic granula. Moreover, its expression significantly correlated with (a) the morphological/histopathological subtype of NSCLC, being more frequent in adenocarcinomas; (b) the grade of tumor differentiation, displaying an increase in low-grade carcinomas; (c) tumor size, occurring predominantly in smaller tumors; (d) the presence of phosphorylated, activated insulin receptor; (e) the median patient age, being present in relatively younger individuals. Furthermore and interestingly, surrounding atypical adenomatous hyperplastic areas and normal alveolar pneumocytes scored insulin-positive in some of the insulin-negative tumors. In addition, PCR exploration for insulin transcripts in some samples positive for immunoreactive insulin was negative, indicating a possibly exogenous origin for the intracellular insulin in our NSCLC cohort. Taken together, our data suggest that an intracellular insulin activity is important for the progression of low-grade human lung adenocarcinomas.

HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome.

Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDHbright cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.

BCL-XL overexpression promotes tumor progression-associated properties.

By using human melanoma and glioblastoma cell lines and their derivative BCL-XL overexpressing clones, we investigated the role of BCL-XL in aggressive features of these two tumor histotypes. We found that in both models, BCL-XL overexpression increased in vitro cell migration and invasion and facilitated tumor cells to form de novo vasculogenic structures. Furthermore, BCL-XL overexpressing cells exhibited higher tumors sphere formation capacity and expressed higher levels of some stem cell markers, supporting the concept that BCL-XL plays essential roles in the maintenance of cancer stem cell phenotype. BCL-XL expression reduction by siRNA, the exposure to a BCL-XL-specific inhibitor and the use of a panel of human melanoma cell lines corroborated the evidence that BCL-XL regulates tumor progression-associated properties. Finally, the vascular markers and the vasculogenic mimicry were up-regulated in the BCL-XL overexpressing xenografts derived from both tumor histotypes. In conclusion, our work brings further support to the understanding of the malignant actions of BCL-XL and, in particular, to the concept that BCL-XL promotes stemness and contributes to the aggressiveness of both melanoma and glioblastoma.

Detection of ATM germline variants by the p53 mitotic centrosomal localization test in BRCA1/2-negative patients with early-onset breast cancer.

BACKGROUND: Variant ATM heterozygotes have an increased risk of developing cancer, cardiovascular diseases, and diabetes. Costs and time of sequencing and ATM variant complexity make large-scale, general population screenings not cost-effective yet. Recently, we developed a straightforward, rapid, and inexpensive test based on p53 mitotic centrosomal localization (p53-MCL) in peripheral blood mononuclear cells (PBMCs) that diagnoses mutant ATM zygosity and recognizes tumor-associated ATM polymorphisms. METHODS: Fresh PBMCs from 496 cancer patients were analyzed by p53-MCL: 90 cases with familial BRCA1/2-positive and -negative breast and/or ovarian cancer, 337 with sporadic cancers (ovarian, lung, colon, and post-menopausal breast cancers), and 69 with breast/thyroid cancer. Variants were confirmed by ATM sequencing. RESULTS: A total of seven individuals with ATM variants were identified, 5/65 (7.7 %) in breast cancer cases of familial breast and/or ovarian cancer and 2/69 (2.9 %) in breast/thyroid cancer. No variant ATM carriers were found among the other cancer cases. Excluding a single case in which both BRCA1 and ATM were mutated, no p53-MCL alterations were observed in BRCA1/2-positive cases. CONCLUSIONS: These data validate p53-MCL as reliable and specific test for germline ATM variants, confirm ATM as breast cancer susceptibility gene, and highlight a possible association with breast/thyroid cancers.

Expectoration of the staple line: a delayed complication after previous lobectomy.

In the literature, bronchopleural fistula after thoracic surgery is usually reported in the perioperative setting; however, the incidence of delayed fistula still remains unknown. We herein report a case of a 54-year old man who was referred to the Emergency Department for dyspnoea and productive cough with expectoration of a whitish 'suture-like' tissue. Two months before, the patient underwent an uneventful right lower lobectomy for squamous-cell lung cancer. A computed tomography scan revealed a para-mediastinal localized pneumothorax (measuring 6 cm in the major axis) starting from a right lower bronchus interruption associated with a middle lobe consolidation. The patient was consequently admitted for respiratory monitoring and underwent a fibre-optic bronchoscopy to assess bronchial integrity: the right lower bronchus was completely dehiscent and connected to an empty pleural closed pouch, with no inflammatory signs or sputum-like effusion. After the procedure, the clinical course was unremarkable with gradual improvement of clinical conditions: in particular, the cough disappeared and no sign of fever, leucocytosis or respiratory failure was observed. Thus, after a multidisciplinary meeting, the patient was discharged 7 days after admission and re-evaluated monthly. Six months after diagnosis, the patient was symptom-free with complete closure of the fistula on radiological imaging.

Prognostic Score of Long-Term Survival After Surgery for Malignant Pleural Mesothelioma: A Multicenter Analysis.

BACKGROUND: Despite ongoing efforts to improve therapy in malignant pleural mesothelioma, few patients undergoing extrapleural pneumonectomy experience long-term survival (LTS). This study aims to explore predictors of LTS after extrapleural pneumonectomy and to define a prognostic score. METHODS: From January 2000 to December 2010, we retrospectively reviewed clinicopathologic and oncological factors in a multicenter cohort of 468 malignant pleural mesothelioma patients undergoing extrapleural pneumonectomy. LTS was defined as survival longer than 3 years. Associations were evaluated using χ(2), Student's t, and Mann-Whitney U tests. Logistic regression, Cox regression hazard model, and bootstrap analysis were applied to identify outcome predictors. Survival curves were calculated by the Kaplan-Meier method. Receiver operating characteristic analyses were used to estimate optimal cutoff and area under the curve for accuracy of the model. RESULTS: Overall, 107 patients (22.9%) survived at least 3 years. Median overall, cancer-specific, and disease-free survival times were 60 (95% confidence interval [CI], 51 to 69), 63 (95% CI, 54 to 72), and 49 months (95% CI, 39 to 58), respectively. At multivariate analysis, age (odds ratio, 0.51; 95% CI, 0.31 to 0.82), epithelioid histology (odds ratio, 7.07; 95% CI, 1.56 to 31.93), no history of asbestos exposure (odds ratio, 3.13; 95% CI, 1.13 to 8.66), and the ratio between metastatic and resected lymph nodes less than 22% (odds ratio, 4.12; 95% CI, 1.68 to 10.12) were independent predictors of LTS. According to these factors, we created a scoring system for LTS that allowed us to correctly predict overall, cancer-specific, and disease-free survival in the total sample, obtaining two different groups with favorable or poor prognosis (area under the curve, 0.74; standard error, 0.04; p < 0.0001). CONCLUSIONS: Our prognostic model facilitates the prediction of LTS after surgery for malignant pleural mesothelioma and can help to stratify the outcome and, eventually, tailor postoperative treatment.