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Epidemiological studies support the idea that multiple sclerosis (MS) is a multifactorial disease, overlapping genetic, epigenetic, and environmental factors. A better definition of environmental risks is critical to understand both etiology and the sex-related differences of MS. Exposure to endocrine-disrupting compounds (EDCs) fully represents one of these risks. EDCs are natural or synthetic exogenous substances (or mixtures) that alter the functions of the endocrine system. Among synthetic EDCs, exposure to bisphenol A (BPA) has been implicated in the etiology of MS, but to date, controversial data has emerged. Furthermore, nothing is known about bisphenol S (BPS), one of the most widely used substitutes for BPA. As exposure to bisphenols will not disappear soon, it is necessary to clarify their role also in this pathological condition defining their role in disease onset and course in both sexes. In this study, we examined, in both sexes, the effects of perinatal exposure to BPA and BPS in one of the most widely used mouse models of MS, experimental autoimmune encephalomyelitis (EAE). Exposure to bisphenols seemed to be particularly deleterious in males. In fact, both BPA- and BPS-treated males showed anticipation of the disease onset and an increased motoneuron loss in the spinal cord. Overall, BPA-treated males also displayed an exacerbation of EAE course and an increase in inflammation markers in the spinal cord. Analyzing the consequences of bisphenol exposure on EAE will help to better understand the role of both xenoestrogens and endogenous estrogens on the sexually dimorphic characteristics of MS.

Purpose: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non–small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. Experimental Design: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. Results: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti–PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P < 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P < 0.0001) and inhibited by PD-1 blockade (−30% ± 3, P < 0.0001). The intravenous monotherapy with anti–PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. Conclusions: We report first evidence of a novel lymphocyte-independent activity of anti–PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505

Papillary thyroid carcinoma (PTC) is considered an indolent neoplasm but it may demonstrate aggressive behavior. We aimed to identify clinical and pathological characteristics and molecular signatures associated with aggressive forms of PTCs. We selected 43 aggressive PTC cases based on the presence of metastases at the time of diagnosis, the development of distant metastasis during follow-up, and/or biochemical recurrence, and 43 PTC patients that were disease-free upon follow-up, matching them according to age, sex, pT, and pN parameters. Twenty-four pairs (a total of 48 cases) and 6 normal thyroid tissues were studied using targeted mRNA screening of cancer-associated genes employing NanoString nCounter® technology. In general, aggressive PTCs showed distinctive clinical and morphological features. Among adverse prognostic parameters, the presence of necrosis and an increased mitotic index were associated with shorter disease-free and overall survivals. Other parameters associated with shorter disease-free or overall survivals include a lack of tumor capsule, the presence of vascular invasion, tumor-infiltrating lymphocytes, fibrosclerotic changes, age > 55 years, and a high pTN stage. Various pathways were differentially regulated in non-aggressive as compared to aggressive PTC, including the DNA damage repair, the MAPK, and the RAS pathways. In particular, the hedgehog pathway was differentially de-regulated in aggressive PTC as compared to non-aggressive PTC cases, being WNT10A and GLI3 genes significantly up- and down-regulated in aggressive PTC and GSK3B up-regulated in non-aggressive PTC cases. In conclusion, our study revealed specific molecular signatures and morphological features in aggressive PTC that may be useful to predict more aggressive behavior in a subset of PTC patients. These findings may be useful when developing novel, tailored treatment options for these patients.

PD-L1/2 and phospho-p38 MAPK expression in NSCLC cells. Tumor membrane expression of PD-L1 and PD-L2 in NSCLC cell lines was intensely enhanced by treatment with Interferon γ (n=9, PD-L1 p=0.02, PD-L2 p

Treatment with PD-L1s is associated with the up-regulation of expression of key protein coding genes involved in processes of tumorigenesis, EMT, tumor aggressiveness and chemoresistance. Heatmap showing z-scaled DESeq2-normalized expression values of the 14 differentially expressed genes between PD-L1s and untreated cells. Right annotation indicates the mean DESeq2-normalized gene expression values. Top annotations depict the pneumosphere of origin and the treatment. Hierarchical clustering of genes and samples is shown on the left and top, respectively (A). Volcano plot showing the log2 fold change (log2FC) and adjusted p-value values of all genes between PD-L1s and untreated samples. Differentially expressed genes are depicted in red (B).

Double positive OCT3/4-PD1 subsets in the bulk NSCLC cells after cisplatin treatment. OCT3/4 expression preferentially co-localized on PD-1+ NSCLC cells as compared with the PD-1- counterpart (14% vs 0.1%). Representative flow cytometry histograms of OCT3/4 expression are reported in figure.

SUPPLEMENTARY FIGURE 1 Correlation of VIM/CDH1 ratios in different datasets. The list of 35 genes significantly correlated with EMT. Lack of correlation between AKR1B10 mRNA levels and EMT. SUPPLEMENTARY FIGURE 2 AKR1B1 levels are modulated by ZEB1, but not in a direct fashion. SUPPLEMENTARY FIGURE 3 Characteristics of the NSCLC patients stained with immunohistochemistry and impact on survival. Alterations of AKR1B1 levels influence cancer cells growth and cancer stemness. SUPPLEMENTARY FIGURE 4 Impact of SORD knockdown on cancer cells growth and invasion. The combination of the inhibition of polyol pathway gene and cisplatin suppresses in vitro growth. Oxidative consumption rate and ECAR/OCR ratios in polyol pathway-deficient cells. SUPPLEMENTARY FIGURE 5 Co-expression patterns of AKR1B1 and SORD in TCGA datasets. SUPPLEMENTARY FIGURE 6 Effects of treatment with excess glucose on the generation of polyols, on the alteration of EMT and stemness markers, and on the activation of TGF-Beta signaling and TGF-Beta-related genes.

List of differentially expressed genes in all analyzed datasets

Cancer cells alter their metabolism to support their malignant properties. In this study, we report that the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (AKR1B1) strongly correlates with epithelial-to-mesenchymal transition (EMT). This association was confirmed in samples from lung cancer patients and from an EMT-driven colon cancer mouse model with p53 deletion. In vitro, mesenchymal-like cancer cells showed increased AKR1B1 levels, and AKR1B1 knockdown was sufficient to revert EMT. An equivalent level of EMT suppression was measured by targeting the downstream enzyme sorbitol-dehydrogenase (SORD), further pointing at the involvement of the PP. Comparative RNA sequencing confirmed a profound alteration of EMT in PP-deficient cells, revealing a strong repression of TGFβ signature genes. Excess glucose was found to promote EMT through autocrine TGFβ stimulation, while PP-deficient cells were refractory to glucose-induced EMT. These data show that PP represents a molecular link between glucose metabolism, cancer differentiation, and aggressiveness, and may serve as a novel therapeutic target.Significance: A glucose-transforming pathway in TGFβ-driven epithelial-to-mesenchymal transition provides novel mechanistic insights into the metabolic control of cancer differentiation. Cancer Res; 78(7); 1604–18. ©2018 AACR.

Cancer cell dissemination is sustained by cell-autonomous and non-cell-autonomous functions. To disentangle the role of HGF (Hepatocyte Growth Factor) and MET ligand/receptor axis in this complex process, we genetically knocked out the MET gene in cancer cells in which MET is not the oncogenic driver. In this way, we evaluated the contribution of the HGF/MET axis to cancer cell dissemination independently of its direct activities in cells of the tumor microenvironment. The lack of MET expression in MET−/− cells has been proved by molecular characterization. From a functional point of view, HGF stimulation of MET−/− cancer cells was ineffective in eliciting intracellular signaling and in sustaining biological functions predictive of malignancy in vitro (i.e., anchorage-independent growth, invasion, and survival in the absence of matrix adhesion). Cancer cell dissemination was assessed in vivo, evaluating: (i) the ability of MET−/− lung carcinoma cells to colonize the lungs following intravenous injection and (ii) the spontaneous dissemination to distant organs of MET−/− pancreatic carcinoma cells upon orthotopic injection. In both experimental models, MET ablation affects the time of onset, the number, and the size of metastatic lesions. These results define a crucial contribution of the HGF/MET axis to cell-autonomous functions driving the metastatic process.

Case presentation and management A 81-years old female presented to the emergency department of our hospital with dyspnea and chest pain. She had a history of chronic kidney disease and left bundle brunch block (LBBB) never further investigated. Non diagnostic ECG was performed in the setting of pre-existing LBBB. HS-T-Troponin level was significantly elevated (986 ng/L). A transthoracic echocardiogram (TTE) revealed inferior and posterior wall hypokinesis. NSTEMI was diagnosed and coronary angiography via the femoral artery revealed subocclusive stenosis of the right coronary artery (RCA) and 80% stenosis of left anterior descending artery (LAD). Coronary stent positioning was performed for the RCA culprit lesion and staged complete revascularization was planned. Complications After two days, the patient experienced sharp pain in the right groin and a femoral pseudoaneurysm was diagnosed by Ultrasound Color Doppler. Subsequently anemia suggested the development of retroperitoneal hematoma but it was excluded by contrast CT. Unfortunately, the patient developed acute chest pain, wheezing and erythematous pruritic rash 10 minutes after injection of contrast medium agent. The ECG didn't show any new ischemic abnormalities in the setting of pre-existing LBBB. The peak CK- MB level was significantly elevated (87 ng/mL). New onset apical region akinesia and anterior wall hypokinesia were detected on TTE. Acute coronary syndrome (ACS) was suspected consistent with symptoms, new onset wall motion abnormalities and CK-MB elevations. The patient was diagnosed with allergic reaction and coronary vessel spasm secondary to contrast agent injection in pre-existing atherosclerotic plaque. Due to the high probability of an hypersensitivity reaction to the contrast medium agent we did not perform a new coronary angiography to confirm the diagnosis. Promethazine, Urbason and Nitroglicerin were administered with clinical improvement. The next day we observed recovery of the anterior and apex wall motion abnormalities consistent with coronary vessel spasm. Echocardiographic wall motion abnormalities before and after coronary spasm: Take home message We presented the case of an allergic reaction leading to the release of inflammatory mediators responsible of coronary vessel spasm causing acute myocardial infarction. It can also be induced by injection of contrast medium like in the presented case. Therefore, clinicians and radiologist should recognize this rare but critical disease to ensure prevention and appropriate management.

Germline mutations in the tumor suppressor gene BRCA1-associated protein-1 (

Extra-pulmonary neuroendocrine carcinomas (EPNEC) represent a group of rare and heterogenous neoplasms with adverse clinical outcome. Their molecular profile is largely unexplored. Our aim was to investigate if the major transcriptional drivers recently described in high-grade pulmonary neuroendocrine carcinomas characterize distinct molecular and clinical subgroups of EPNEC. Gene expression of ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3, and YAP1 was investigated in a series of 54 EPNEC (including 10 cases with mixed components analyzed separately) and in a group of 48 pulmonary large cell neuroendocrine carcinomas (P-LCNEC). Unsupervised hierarchical cluster analysis classified the whole series into four major clusters. P-LCNEC were classified into two major clusters, the first ASCL1/DLL3/INSM1-high and the second (including four EPNEC) ASCL1/DLL3-low but INSM1-high. The remaining EPNEC cases were sub-classified into two other clusters. The first showed INSM1-high and alternative ASCL1/DLL3 or NEUROD1 high expression. The second was characterized mainly by MYCL1 and YAP1 overexpression. In the ten cases with mixed histology, ASCL1, DLL3, INSM1, and NEUROD1 genes were significantly upregulated in the neuroendocrine component. Higher gene-expression levels of NOTCH1 and INSM1 were associated with lower pT stage and negative nodal status. Low INSM1 gene expression was associated with shorter overall survival in the entire case series (p = 0.0017) and with a trend towards significance in EPNEC, only (p = 0.06). In conclusion, our results show that EPNEC possess distinct neuroendocrine-lineage-specific transcriptional profiles; moreover, low INSM1 gene expression represents a novel potential unfavorable prognostic marker in high-grade NECs including those in extra-pulmonary location.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically “neoplastic” lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

A 13-year-old boy complained of shortness of his left arm with the desire for lengthening. The X-Ray showed the enlarged ellipsoid shaped humeral epiphysis in varus position and irregular joint surface. The MRI documented a distorted bone structure involving the complete epiphysis, overgrowth of the tuberosities and partial closure of the physis. MR-angiography revealed normally appearing vessel formation; however, a biopsy was recommended to rule out a vascular malformation. A tru-cut needle biopsy confirmed the diagnosis of enchondromatous changes. As the patient felt strongly disturbed by the shortness of his arm, lengthening was performed using the fully inserted magnetic driven PreciceR nail. Conclusion: This case is of interest as epiphysial enchondromas are rare and complete involvement of the proximal humeral epiphysis causing growth deficiency to our knowledge has never been described before. Lengthening using a device penetrating the cartilaginous region is debatable; however, so far, sarcomatous dedifferentiation in epiphysial enchondromas has not been described.

Pediatric neuroblastoma is responsible for approximately 8–10% of pediatric tumors, and it is one of the leading causes of tumor-related deaths in children. Although significant progress has been made in the characterization of neuroblastoma in recent years, the mechanisms influencing the prognosis of neuroblastoma patients remain largely unknown. Our aim was to investigate if the major neuroendocrine-associated transcriptional drivers, including ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3 and YAP1 are correlated with specific clinical and pathological characteristics. We selected a retrospective series of 46 primary pediatric neuroblastoma, composed of 30 treatment-naïve and 16 post-chemotherapy cases. Gene expression levels were explored by means of quantitative real-time PCR. An increased expression of NOTCH1 (p = 0.005), NEUROD1 (p = 0.0059), and YAP1 (p = 0.0008) was found in stage IV tumors, while the highest levels of MYCL1 and ASCL1 were seen in stages IVS and III, respectively (p = 0.0182 and p = 0.0134). A higher level of NOTCH1 (p = 0.0079) and YAP1 (p = 0.0026) was found in cases with differentiating morphology, while high mitosis-karyorrhexis index cases demonstrated significantly lower levels of POU2F3 (p = 0.0277). High expression of NOTCH1 (p = 0.008), NEUROD1 (p = 0.026), INSM1 (p = 0.010), and YAP1 (p = 0.005) together with stage IV (p = 0.043) was associated with shorter disease-free survival. In summary, our data indicate that the assessment of gene expression levels of neuroendocrine-lineage transcription factors might help to identify neuroblastoma patients with the risk of relapse.

International audience; Loire constitutes, in its western part, a major element in the structuring of the landscape and the territories that it crosses in historical periods. This observation, which can be applied to the Iron Age according to the texts of Strabo and Caesar in particular, has only really started to be documented by archeology in recent years. The aim of this contribution is therefore to bring together largely unpublished documentation in order to provide information on three main questions : Development and management of the watercourse (bridges, fords, banks, wrecks) ; The status and organization of sites related to the Loire, in a 50 km wide strip; Cultural entities crossed by the river. Bringing together the results of numerous preventive archaeological interventions and recent academic work, the study demonstrates the Loire's significant attraction for the peoples concerned and its obvious role for long-distance exchanges. It also highlights the preeminent role of the Gallic populations in the human appropriation of the valley.; La Loire constitue, dans sa portion occidentale, un élément majeur de la structuration du paysage et des territoires qu’elle traverse aux périodes historiques. Ce constat, que l’on peut appliquer à l’âge du Fer d’après les textes de Strabon et de César notamment, ne commence véritablement à être documenté par l’archéologie que depuis quelques années. Cette contribution a donc pour objectif de rassembler une documentation en grande partie inédite, afin de renseigner trois questions principales : Les aménagements et la gestion du cours d’eau (ponts, gués, berges, épaves) ; Le statut et l’organisation des sites en relation avec la Loire, dans une bande de 50 km de large ; Les entités culturelles traversées par le fleuve. Réunissant les résultats de nombreuses interventions d’archéologie préventives et de travaux universitaires récents, l’étude démontre l’attraction importante de la Loire pour les peuples concernés et son rôle manifeste pour les échanges à longue distance. Elle permet également de mettre en évidence le rôle prééminent des populations gauloises dans l’appropriation humaine du val.

Neurogenesis, the regulation of cellular proliferation and differentiation in the developing nervous system, is the process that underlies the diversity of size and cell type found in animal nervous systems. Our understanding of how this process has evolved is limited because of the lack of high resolution data and live-imaging methods across species. The retina is a classic model for the study of neurogenesis in vertebrates and live-imaging of the retina has shown that during development, progenitor cells are organized in a pseudostratified neuroepithelium and nuclei migrate in coordination with the cell cycle along the apicobasal axis of the cell, a process called interkinetic nuclear migration. Eventually cells delaminate and differentiate within the boundaries of the epithelium. This process has been considered unique to vertebrates and thought to be important in maintaining organization during the development of a complex nervous system. Coleoid cephalopods, including squid, cuttlefish and octopus, have the largest nervous system of any invertebrate and convergently-evolved camera-type eyes, making them a compelling comparative system to vertebrates. Here we have pioneered live-imaging techniques to show that the squid, Doryteuthis pealeii, displays cellular mechanisms during cephalopod retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo interkinetic nuclear migration until they exit the cell cycle, we identify retinal organization corresponding to progenitor, post-mitotic and differentiated cells, and we find that Notch signaling regulates this process. With cephalopods and vertebrates having diverged 550 million years ago, these results suggest that mechanisms thought to be unique to vertebrates may be common to highly proliferative neurogenic primordia contributing to a large nervous system.

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM.

Lung cancer represents the leading cause of cancer-related mortality worldwide, with NSCLC being the most common type. Two-thirds of NSCLC patients harbor oncogenic driver alterations, among which EGFR activating mutations and ALK rearrangements occur in 16% and 5% of cases, respectively. However, in both genetic backgrounds the benefit of molecular therapies, including next generation Tyrosine Kinase Inhibitors (TKIs), is temporally limited with therapy evasion representing a major roadblock against cancer eradication. Patient Derived Organoids (PDOs) have raised a tremendous interest in cancer research for their ability to preserve the original tumoral architecture, including its microenvironment, at both the genomic and functional level. Notably, PDOs can be stabilized from a very limited amount of starting material, such as needle biopsies from local and distal metastases, including lesions originated upon molecular therapy relapse. The possibility to perform pharmacogenomic screening and functional analyses renders PDOs an ideal tool for co-clinical studies. Thereby, we have systematically collected 87 oncogene-addicted lung cancer specimens, among which 48 were suitable for 3D cultures in terms of tumor cell number and viability. We successfully established 15 long-term PDO cultures that maintain the proliferation capability throughout passages, and obtained a subset of corresponding 2D cell lines. Histological and immunohistochemical analyses revealed that PDOs faithfully recapitulate the features of the original neoplastic lesions. Moreover, targeted molecular annotation confirmed that our PDOs retained the somatic oncogene alterations of the matching tumors. In addition, drug sensitivity tests and functional experiments have demonstrated the ability of PDOs to reproduce patient response to targeted therapies, indicating the translational relevance of our platform. Furthermore, we exploited organoids to study the molecular circuits responsible for tumor cell evasion to targeted therapy, identifying bypass track signaling as critical for the emergence of drug resistance. Finally, we tested the therapeutic efficacy of drug treatments based on the combination of early and next generations TKIs. Overall, our data highlight the translational relevance of PDO interrogation in clinical oncology, especially for patients who relapse to different lines of therapy. We provide evidence that in oncogene-addicted tumors bypass track signaling is a common non-genetic mechanism of resistance to last generation TKIs. Thus, the correct co-administration of different molecules and/or the use of earlier-generation inhibitors upon progression may improve treatment strategies, potentially impacting on patient’s benefit and clinical outcome. Citation Format: Francesca Picca, Francesca Bersani, Fabrizio Tabbo', Francesca Napoli, Paolo Bironzo, Angela Listi', Francesco Passiglia, Marco Volante, Giorgio Vittorio Scagliotti, Luisella Righi, SIlvia Novello, Riccardo Taulli. Patient-derived organoids from oncogene-addicted lung cancers to define molecular circuits involved in drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6208.

Simple Summary The marine drug lurbinectedin revealed an unprecedented efficacy against patient-derived malignant pleural mesothelioma cells, regardless of the histological type and the BAP1 mutation status. By inducing strong DNA damages, it dramatically arrested cell cycle progression and induced apoptosis. These results may be translated into the use of lurbinectedin as an effective agent for malignant pleural mesothelioma patients. Abstract Background: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer generally diagnosed at an advanced stage and characterized by a poor prognosis. The absence of alterations in druggable kinases, together with an immune-suppressive tumor microenvironment, limits the use of molecular targeted therapies, making the treatment of MPM particularly challenging. Here we investigated the in vitro susceptibility of MPM to lurbinectedin (PM01183), a marine-derived drug that recently received accelerated approval by the FDA for the treatment of patients with metastatic small cell lung cancer with disease progression on or after platinum-based chemotherapy. Methods: A panel of primary MPM cultures, resembling the three major MPM histological subtypes (epithelioid, sarcomatoid, and biphasic), was characterized in terms of BAP1 status and histological markers. Subsequently, we explored the effects of lurbinectedin at nanomolar concentration on cell cycle, cell viability, DNA damage, genotoxic stress response, and proliferation. Results: Stabilized MPM cultures exhibited high sensitivity to lurbinectedin independently from the BAP1 mutational status and histological classification. Specifically, we observed that lurbinectedin rapidly promoted a cell cycle arrest in the S-phase and the activation of the DNA damage response, two conditions that invariably resulted in an irreversible DNA fragmentation, together with strong apoptotic cell death. Moreover, the analysis of long-term treatment indicated that lurbinectedin severely impacts MPM transforming abilities in vitro. Conclusion: Overall, our data provide evidence that lurbinectedin exerts a potent antitumoral activity on primary MPM cells, independently from both the histological subtype and BAP1 alteration, suggesting its potential activity in the treatment of MPM patients.

The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. Here, we show HSPD1 (HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients’ prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on OXPHOS and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NCSLC.SignificanceHSPD1 elimination or disruption interferes with NSCLC metabolic activity causing a strong OXPHOS-dependent energetic breakdown, which the cancer cells fail to overcome, highlighting HSPD1 as a potential theranostic marker for improving lung cancer therapy.

Since their discovery, microRNAs (miRNAs) have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity for in vitro and in vivo applications could lead to a better understanding of their biological role in physiology and disease.

The molecular mechanisms of adrenocortical carcinoma development are incompletely defined. De-regulation of cellular-to-extracellular matrix interactions and angiogenesis appear among mechanisms associated to the malignant phenotype. Our aim was to investigate, employing PCR-based array profiling, 157 molecules involved in cell-to-matrix interactions and angiogenesis in a frozen series of 6 benign and 6 malignant adrenocortical neoplasms, to identify novel pathogenetic markers. In 14 genes, a significant dysregulation was detected in adrenocortical carcinomas as compared to adenomas, most of them being downregulated. Three exceptions—hyaluronan synthase 1 (HAS-1), laminin α3 and osteopontin genes—demonstrated an increased expression in adrenocortical carcinomas of 4.46, 4.23 and 20.32-fold, respectively, and were validated by immunohistochemistry on a series of paraffin-embedded tissues, including 20 adenomas and 73 carcinomas. Osteopontin protein, absent in all adenomas, was expressed in a carcinoma subset (25/73) (p = 0.0022). Laminin α3 and HAS-1 were mostly expressed in smooth muscle and endothelial cells of the vascular network of both benign and malignant adrenocortical tumors. HAS-1 was also detected in tumor cells, with a more intense pattern in carcinomas. In this group, strong expression was significantly associated with more favorable clinicopathological features. These data demonstrate that cell-to-matrix interactions are specifically altered in adrenocortical carcinoma and identify osteopontin and HAS-1 as novel potential diagnostic and prognostic biomarkers, respectively, in adrenal cortical tumors.

Simple Summary Tumor immune microenvironment is an important structural component of malignant pleural mesothelioma that contributes to disease growth support and progression. Its study and pathological characterization are important tools to find new biomarkers for advanced therapeutic strategies. Abstract Malignant pleural mesothelioma (MPM) is a rare and highly aggressive disease that arises from pleural mesothelial cells, characterized by a median survival of approximately 13–15 months after diagnosis. The primary cause of this disease is asbestos exposure and the main issues associated with it are late diagnosis and lack of effective therapies. Asbestos-induced cellular damage is associated with the generation of an inflammatory microenvironment that influences and supports tumor growth, possibly in association with patients’ genetic predisposition and tumor genomic profile. The chronic inflammatory response to asbestos fibers leads to a unique tumor immune microenvironment (TIME) composed of a heterogeneous mixture of stromal, endothelial, and immune cells, and relative composition and interaction among them is suggested to bear prognostic and therapeutic implications. TIME in MPM is known to be constituted by immunosuppressive cells, such as type 2 tumor-associated macrophages and T regulatory lymphocytes, plus the expression of several immunosuppressive factors, such as tumor-associated PD-L1. Several studies in recent years have contributed to achieve a greater understanding of the pathogenetic mechanisms in tumor development and pathobiology of TIME, that opens the way to new therapeutic strategies. The study of TIME is fundamental in identifying appropriate prognostic and predictive tissue biomarkers. In the present review, we summarize the current knowledge about the pathological characterization of TIME in MPM.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Since their discovery, microRNAs (miRNA)s have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs’ endogenous levels and activity could lead to a better understanding of their biological role in physiology and disease.

Objective To evaluate effectiveness, in terms of patient-reported outcome measures, of platelet-rich plasma (PRP) injections for knee osteoarthritis compared to placebo and other intraarticular treatments. Design PubMed, Cochrane Library, Scopus, Embase, Web of Science, as well as the gray literature were searched on January 17, 2020. Randomized controlled trials (RCTs) comparing PRP injections with placebo or other injectable treatments, in any language, on humans, were included. Risk of bias was assessed following the Cochrane guidelines; quality of evidence was graded using the GRADE guidelines. Results Thirty-four RCTs, including 1403 knees in PRP groups and 1426 in control groups, were selected. WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) score favored PRP, with a statistically and clinically significant difference versus placebo at 12-month follow-up ( P = 0.02) and versus HA (hyaluronic acid) at 6-month ( P < 0.001) and 12-month ( P < 0.001) follow-ups. A clinically significant difference favoring PRP versus steroids was documented for VAS (Visual Analogue Scale) pain ( P < 0.001), KOOS (Knee Injury and Osteoarthritis Outcome Score) pain ( P < 0.001), function in daily activities ( P = 0.001), and quality of life ( P < 0.001) at 6-month follow-up. However, superiority of PRP did not reach the minimal clinically important difference for all outcomes, and quality of evidence was low. Conclusions The effect of platelet concentrates goes beyond its mere placebo effect, and PRP injections provide better results than other injectable options. This benefit increases over time, being not significant at earlier follow-ups but becoming clinically significant after 6 to 12 months. However, although substantial, the improvement remains partial and supported by low level of evidence. This finding urges further research to confirm benefits and identify the best formulation and indications for PRP injections in knee OA.

Aim: To validate the prognostic role of a panel of genes previously uncovered by our group to be specific targets of miRNAs differentially expressed in lung carcinoids with aggressive pathological features. Methods: Four genes, namely, cyclic AMP response element binding protein-1 (CREBP1), activin A receptor type 2B (ACVR2B), LIM homeobox 2 (LHX2), and Krüppel-like factor 12 (KLF12), were identified in a previous study by our group using in silico analysis to be regulated by 3 miRNAs (miR-409-3p, miR-409-5p, and miR-431-5p) that were shown to be downregulated in aggressive lung carcinoids. These genes were analyzed using real-time PCR in a cohort of 102 lung carcinoids. Fifty high-grade lung carcinomas served as control group. Their expression was correlated with the expression of miR-409-3p, miR-409-5p, and miR-431-5p and with clinical pathological parameters and disease-free survival. Results: The expression of all but CREBP1 gene was significantly different between lung carcinoids and high-grade neuroendocrine carcinomas. ACVR2B and LHX2 were significantly inversely correlated with miR-409-3p and miR-409-5p. High levels of ACVR2B and LHX2 were significantly associated with atypical histotype, high tumor grade, and higher proliferation Ki-67 index (all p < 0.05). Low levels of KLF12 were significantly associated with the presence of necrosis and positive nodal status (all p < 0.05). Finally, low KLF12 expression was associated with shorter disease-free survival in lung carcinoids as a whole and in atypical carcinoids, only (all p < 0.001). Conclusions: ACVR2B, LHX2, and KFL12 are novel potential biomarkers associated with aggressive features in lung carcinoids.

Malignant pleural mesothelioma (MPM) is a tumor with high chemoresistance and poor prognosis. MPM-initiating cells (ICs) are known to be drug resistant, but it is unknown if and how stemness-related pathways determine chemoresistance. Moreover, there are no predictive markers of IC-associated chemoresistance. Aim of this work is to clarify if and by which mechanisms the chemoresistant phenotype of MPM IC was due to specific stemness-related pathways. We generated MPM IC from primary MPM samples and compared the gene expression and chemo-sensitivity profile of IC and differentiated/adherent cells (AC) of the same patient. Compared to AC, IC had upregulated the drug efflux transporter ABCB5 that determined resistance to cisplatin and pemetrexed. ABCB5-knocked-out (KO) IC clones were resensitized to the drugs in vitro and in patient-derived xenografts. ABCB5 was transcriptionally activated by the Wnt/GSK3β/β-catenin/c-myc axis that also increased IL-8 and IL-1β production. IL-8 and IL-1β-KO IC clones reduced the c-myc-driven transcription of ABCB5 and reacquired chemosensitivity. ABCB5-KO clones had lower IL-8 and IL-1β secretion, and c-myc transcriptional activity, suggesting that either Wnt/GSK3β/β-catenin and IL-8/IL-1β signaling drive c-myc-mediated transcription of ABCB5. ABCB5 correlated with lower time-to-progression and overall survival in MPM patients treated with cisplatin and pemetrexed. Our work identified multiple autocrine loops linking stemness pathways and resistance to cisplatin and pemetrexed in MPM IC. ABCB5 may represent a new target to chemosensitize MPM IC and a potential biomarker to predict the response to the first-line chemotherapy in MPM patients.

Cancer cells alter their metabolism to support their malignant properties. In this study, we report that the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (AKR1B1) strongly correlates with epithelial-to-mesenchymal transition (EMT). This association was confirmed in samples from lung cancer patients and from an EMT-driven colon cancer mouse model with p53 deletion. In vitro, mesenchymal-like cancer cells showed increased AKR1B1 levels, and AKR1B1 knockdown was sufficient to revert EMT. An equivalent level of EMT suppression was measured by targeting the downstream enzyme sorbitol-dehydrogenase (SORD), further pointing at the involvement of the PP. Comparative RNA sequencing confirmed a profound alteration of EMT in PP-deficient cells, revealing a strong repression of TGFb signature genes. Excess glucose was found to promote EMT through autocrine TGFb stimulation, while PP-deficient cells were refractory to glucose-induced EMT. These data show that PP represents a molecular link between glucose metabolism, cancer differentiation, and aggressiveness, and may serve as a novel therapeutic target. This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nuremberg, the Deutsche Krebshilfe grant number 70112536, the IALSC Lung Cancer Young Investigator Award (to P. Ceppi), and by the Clinical Research GroupKFO262 funded by the German Research Foundation. This work was partially presented at the American Association for Cancer Research 2017 annual meeting. Special thanks to Dr. H. Wurdak (University of Leeds) for critical discussion.

Thymidylate synthase (TS) is a fundamental enzyme of nucleotide metabolism and one of the oldest anti-cancer targets. Beginning from the analysis of gene array data from the NCI-60 panel of cancer cell lines, we identified a significant correlation at both gene and protein level between TS and the markers of epithelial-to-mesenchymal transition (EMT), a developmental process that allows cancer cells to acquire features of aggressiveness, like motility and chemoresistance. TS levels were found to be significantly augmented in mesenchymal-like compared to epithelial-like cancer cells, to be regulated by EMT induction, and to negatively correlate with micro-RNAs (miRNAs) usually expressed in epithelial-like cells and known to actively suppress EMT. Transfection of EMT-suppressing miRNAs reduced TS levels, and a specific role for miR-375 in targeting the TS 3'-untranslated region was identified. A particularly relevant association was found between TS and the powerful EMT driver ZEB1, the shRNA-mediated knockdown of which up-regulated miR-375 and reduced TS cellular levels. The TS-ZEB1 association was confirmed in clinical specimens from lung tumours and in a genetic mouse model of pancreatic cancer with ZEB1 deletion. Interestingly, TS itself appeared to have a regulatory role in EMT in cancer cells, as TS knockdown could directly reduce the EMT phenotype, the migratory ability of cells, the expression of stem-like markers, and chemoresistance. Taken together, these data indicate that the TS enzyme is functionally linked with EMT and cancer differentiation, with several potential translational implications. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction A comprehensive analysis of the immune cell infiltrate collected from pleural fluid and from biopsy specimens of malignant pleural mesothelioma (MPM) may contribute to understanding the immune-evasion mechanisms related to tumor progression, aiding in differential diagnosis and potential prognostic stratification. Until now such approach has not routinely been verified. Methods We enrolled 275 patients with an initial clinical diagnosis of pleural effusion. Specimens of pleural fluids and pleural biopsy samples used for the pathologic diagnosis and the immune phenotype analyses were blindly investigated by multiparametric flow cytometry. The results were analyzed using the Kruskal-Wallis test. The Kaplan-Meier and log-rank tests were used to correlate immune phenotype data with patients’ outcome. Results The cutoffs of intratumor T-regulatory (>1.1%) cells, M2-macrophages (>36%), granulocytic and monocytic myeloid-derived suppressor cells (MDSC; >5.1% and 4.2%, respectively), CD4 molecule–positive (CD4+) programmed death 1–positive (PD-1+) (>5.2%) and CD8+PD-1+ (6.4%) cells, CD4+ lymphocyte activating 3–positive (LAG-3+) (>2.8% ) and CD8+LAG-3+ (>2.8%) cells, CD4+ T cell immunoglobulin and mucin domain 3–positive (TIM-3+) (>2.5%), and CD8+TIM-3+ (>2.6%) cells discriminated MPM from pleuritis with 100% sensitivity and 89% specificity. The presence of intratumor MDSC contributed to the anergy of tumor-infiltrating lymphocytes. The immune phenotype of pleural fluid cells had no prognostic significance. By contrast, the intratumor T-regulatory and MDSC levels significantly correlated with progression-free and overall survival, the PD-1+/LAG-3+/TIM-3+ CD4+ tumor-infiltrating lymphocytes correlated with overall survival. Conclusions A clear immune signature of pleural fluids and tissues of MPM patients may contribute to better predict patients’ outcome.

Purpose: During anterior cruciate ligament (ACL) injury, the large external forces responsible for ligament rupture cause a violent impact between tibial and femoral articular cartilage, which is transferred to bone resulting in bone bruise detectable at MRI. Several aspects remain controversial and await evidence on how this MRI finding should be managed while addressing the ligament lesion. Thus, the aim of the present review was to document the evidence of all available literature on the role of bone bruise associated with ACL lesions. Methods: A systematic review of the literature was performed on bone bruise associated with ACL injury. The search was conducted in September 2017 on three medical electronic databases: PubMed, Web of Science, and the Cochrane Collaboration. Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines were used. Relevant articles were studied to investigate three main aspects: prevalence and progression of bone bruise associated with ACL lesions, its impact on the knee in terms of lesion severity and joint degeneration progression over time and, finally, the influence of bone bruise on patient prognosis in terms of clinical outcome. Results: The search identified 415 records and, after an initial screening according to the inclusion/exclusion criteria, 83 papers were used for analysis, involving a total of 10,047 patients. Bone bruise has a high prevalence (78% in the most recent papers), with distinct patterns related to the mechanism of injury. This MRI finding is detectable only in a minority of cases the first few months after trauma, but its presence and persistence have been correlated to a more severe joint damage that may affect the degenerative progression of the entire joint, with recent evidence suggesting possible effects on long-term clinical outcome. Conclusion: This systematic review of the literature documented a growing interest on bone bruise associated with ACL injury, highlighting aspects which could provide to orthopaedic surgeons evidence-based suggestions in terms of clinical relevance when dealing with patients affected by bone bruise following ACL injury. However, prospective long-term studies are needed to better understand the natural history of bone bruise, identifying prognostic factors and targets of specific treatments that should be developed in light of the overall joint derangements accompanying ACL lesions. Levels of evidence: IV, Systematic review of level I–IV studies.

Purpose: To compare outcomes after surgery versus nonsurgical treatment in the management of primary lateral patellar dislocation (LPD) through a meta-analysis of randomized controlled trials (RCTs) in terms of redislocation rate and clinical outcome, investigating both short-term (6 years). Methods: A systematic search of the literature was performed in November 2018. Risk of bias and quality of evidence were evaluated according to the Cochrane guidelines. RCTs investigating differences between surgery and nonsurgical treatment in primary LPD were included. The outcomes evaluated were redislocation rate, reinterventions, and Kujala score at short-, mid-, and long-term follow-up, with subanalyses for the pediatric population. Results: We included 510 patients from 10 RCTs in the meta-analysis. Redislocation rate was 0.40 (0.25 to 0.66; P < .001) and 0.58 (0.29 to 1.15; P = .12) at the short- and mid-term follow-ups, respectively, and the risk ratio for the need for further operations at 6 to 9 months' follow-up was 0.14 (0.02 to 1.03; P = .05), all favoring surgery. Concerning the Kujala score, an advantage of the surgical approach of 10.2 points (1.6 to 18.7; P = .02) at short-term follow-up was seen, whereas long-term follow-up results were similar between the groups. The subanalysis of the pediatric population at heterogeneous follow-up confirmed a lower risk of recurrence in surgery, with a risk ratio of 0.60 (0.26 to 1.37; P = .22), although not significant. Conclusion: The literature documents a low number of high-level trials. The meta-analysis of RCTs underlined that the redislocation rate is higher with the nonsurgical approach compared with the surgical one. Moreover, when looking at the clinical outcome, more favorable findings were found with the surgical approach up to 6 years, whereas results seems to be similar at a longer follow-up after either surgical or nonsurgical treatment of primary LPD. Level of evidence: II, meta-analysis of level I and level II randomized clinical trials.

Androgen deprivation therapy (ADT) is the standard of care for metastatic prostate cancer and initially induces tumor regression, but invariably results in castration-resistant prostate cancer through various mechanisms, incompletely discovered. Our aim was to analyze the dynamic modulation, determined by ADT, of the expression of selected genes involved in the pathogenesis and progression of prostate cancer (TMPRSS2:ERG, WNT11, SPINK1, CHGA, AR, and SPDEF) using real-time polymerase chain reaction in a series of 59 surgical samples of prostate carcinomas, including 37 cases preoperatively treated with ADT and 22 untreated cases, and in 43 corresponding biopsies. The same genes were analyzed in androgen-deprived and control LNCaP cells. Three genes were significantly up-modulated (WNT11 and AR) or down-modulated (SPDEF) in patients treated with ADT versus untreated cases, as well as in androgen-deprived LNCaP cells. The effect of ADT on CHGA gene up-modulation was almost exclusively detected in cases positive for the TMPRSS2:ERG fusion. The correlation between biopsy and surgical samples was poor for most of the tested genes. Gene expression analysis of separate tumor areas from the same patient showed an extremely heterogeneous profile in the 6 tested cases (all untreated). In conclusion, our results strengthened the implication of ADT in promoting a prostate cancer aggressive phenotype and identified potential biomarkers, with special reference to the TMPRSS2:ERG fusion, which might favor the development of neuroendocrine differentiation in hormone-treated patients. However, intratumoral heterogeneity limits the use of gene expression analysis as a potential prognostic or predictive biomarker in patients treated with ADT.

Background: O6-methylguanine-methyltransferase (MGMT) is a key enzyme for the DNA repair machinery strongly associated with response to alkylating agents in different tumors. Data on its expression and related clinical impact in neuroendocrine tumors are limited to the gastro-entero-pancreatic system, with controversial results in terms of prognostic or predictive value. In lung carcinoids, although clinical efficacy of alkylating agents has been shown in small studies, very few data to date are available on MGMT status. Objective: To assess MGMT status in lung carcinoids using multiple assays and to compare data with major clinical and pathological features. Methods: A retrospective series of 95 lung carcinoids and 51 control cases of high-grade neuroendocrine lung carcinomas was analyzed for MGMT promoter methylation, MGMT gene expression, and MGMT protein expression using pyrosequencing, quantitative real-time PCR, and immunohistochemistry, respectively. Results: MGMT protein expression was inversely correlated with MGMT promoter methylation and positively with MGMT gene expression. MGMT promoter methylation progressively increased from carcinoids to high-grade carcinomas. In the carcinoid group, decreased MGMT gene expression was significantly associated with aggressive features (atypical histotype, grade G2, larger tumor size, higher T stage, and positive nodal status) but not with survival. MGMT promoter methylation was associated with lower stage and negative nodal status. Conclusions: Our study investigated MGMT status in a large series of lung carcinoids in the attempt to move forward a rational use of alkylating agents in these tumors. Interestingly, low MGMT gene expression defines a subgroup of lung carcinoids with aggressive features.