Lung Carcinoid Research Articles

Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia: A Clinical Case.

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare pulmonary condition characterized by diffuse proliferation of neuroendocrine cells in the epithelium of the bronchial wall. DIPNECH may be easily missed in daily clinical practice and diagnosis is often delayed, which may impair prognosis since this condition is considered a pre-invasive lesion for lung carcinoid tumours. We report a clinical case of DIPNECH in order to discuss the diagnostic and therapeutic approach for this entity, the management of which is not yet well established in the literature.LEARNING POINTSDIPNECH is a poorly understood lung condition characterized histologically by diffuse proliferation of pulmonary neuroendocrine cells in the bronchial wall, clinically by obstructive respiratory symptoms and radiologically by small airway disease features and pulmonary nodules.DIPNECH is considered to be a preneoplastic lesion within the spectrum of pulmonary neuroendocrine tumours.Treatment is usually guided by the symptoms and prognosis is highly variable.

Proposal of a Panel of Genes Identified by miRNA Profiling as Candidate Prognostic Biomarkers in Lung Carcinoids

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.

Ectopic Cushing’s Syndrome as the Initial Presentation of Pulmonary Carcinoid Tumor Managed by Etomidate: A Case Report

Ectopic increase of adrenocorticotropic hormone (ACTH) or ectopic Cushing syndrome is a rare initial presentation of small cell carcinoma and carcinoid tumor of the lung. Here, we reported a 36-year-old male with atypical symptoms of Cushing syndrome managed by Etomidate that had a central pulmonary nodule, and pathologic evaluation of pulmonary lobectomy revealed the diagnosis of well-differentiated, low-grade typical carcinoids of lung. © 2019Tehran University of Medical Sciences. All rights reserved. Acta MedIran 2019;57(7):464-467.

SATB2 protein expression by immunohistochemistry is a sensitive and specific marker of appendiceal and rectosigmoid well differentiated neuroendocrine tumours.

Neuroendocrine neoplasms (NNs) range from well to poorly differentiated and indolent to highly aggressive. The site of origin in metastatic NNs has therapeutic and prognostic implications. SATB2 is a transcriptional regulator involved in osteoblastic and neuronal differentiation and is a sensitive and specific marker of colorectal epithelium. This study aimed to evaluate the expression of SATB2 in NNs from various primary sites and its utility as a marker in determining the site of origin of these neoplasms. SATB2 immunohistochemistry was performed on 266 NNs, including lung small cell carcinomas (n=39) and carcinoids (n=30), bladder (n=21) and prostate (n=31) small cell carcinomas, and gastrointestinal (GI)/pancreatic NNs of various primary sites (n=145) consisting of well-differentiated neuroendocrine tumours (WDNET)s (n=124) and poorly differentiated neuroendocrine carcinomas (PDNEC)s (n=21). SATB2 was expressed in prostatic (10 of 31, 32%) and bladder (eight of 21, 38%) small cell carcinomas, lung carcinoid tumours (one of 30, 3%), and lung small cell carcinomas (eight of 39, 21%). Among primary GI NNs, SATB2 was expressed in 37 of 124 (30%) WDNETs and four of 21 (19%) PDNECs. Of the former, 15 of 15 (100%) rectal/rectosigmoid and 22 of 22 (100%) appendiceal neoplasms expressed SATB2. Using receiver operator characteristic analysis, SATB2 was a sensitive and specific marker for rectal (100.0%, 80.0%) and appendiceal (100.0%, 84.5%) WDNETs, respectively. In summary, SATB2 is a sensitive and specific marker for rectal/rectosigmoid and appendiceal WDNETs, and may represent a useful diagnostic tool when these sites of origin are considered in the differential diagnosis.

Cushing´s syndrome: high aggressiveness of low-grade tumors

Cushing´s syndrome is caused by prolonged exposition supraphysiological to endogenous or exogenous cortisol. Ectopic production of adrenocorticotropic hormone by lung carcinoid tumors is relatively rare. Most documented cases have been reported individually. This rare neoplasm low grade that may secrete adrenocorticotropin (ACTH) leading to rapid development of hypercortisolism which is the main mode of discovery, can be a real aggressive form. This report shows a high aggressiveness of this endocrine neoplasia, wich was marked on the general, morphological, bone and psychiatric level. The trivialization of clinical signs had caused the delay in diagnosis with irreparable bone consequences.

Integrative genomic characterization identifies molecular subtypes of lung carcinoids

Integrative genomic characterization identifies molecular subtypes of lung carcinoids

The role of surgery for atypical bronchopulmonary carcinoid tumor: Development and validation of a model based on Surveillance, Epidemiology, and End Results (SEER) database

ObjectivesThe rarity of atypical carcinoid (AC) of lung and the lack of prospective clinical trials lead to limited knowledge of its biology, treatment information and prognosis. The current study analyzed AC patients from the Surveillance, Epidemiology, and End Results (SEER) database to better understand the clinical characteristics of this disease and build a prognostic nomogram for clinical practice. Materials and methodsA total of 507 AC patients with pathological confirmation from SEER database were performed with univariate Cox regression analyses for both overall survival (OS) and lung cancer specific survival (LCSS) analyses. Of the 507 observations, 464 were used in the multivariable Cox proportional hazards model as training cohort of new nomogram. A new nomogram was constructed based on the training cohort and validated by an external validation cohort to predict the 3-, 5- and 10-year OS of ACs. The accuracy and clinical practicability were separately tested by Harrell's C-indexes, calibration plots and decision curve analyses (DCA). ResultsLobectomy and segmental resection were found to be protective factors for AC patients. Age, primary tumor size, N stage, M stage, surgery and regional lymph nodes examination were shown as significant prognostic factors in Cox regression analyses and included in the nomogram as predictors. The C-index in the training cohort for 3-, 5-, and 10-year OS were 0.722, 0.737 and 0.712, respectively. The internal and external calibration plots for predictions of the 3-, 5-, and 10-year OS were in excellent agreement. An online webserver was built based on the proposed nomogram for convenient clinical use. ConclusionAC patients with lobectomy or segmental resection tended to have better OS and LCSS. A nomogram was constructed and validated to predict the OS for AC patients and to provide accurate and individualized survival predictions.

68Ga-DOTATOC PET/CT in detecting neuroendocrine tumours responsible for initial or recurrent paraneoplastic Cushing's syndrome.

Paraneoplastic Cushing's syndrome (PCS) is frequently caused by neuroendocrine tumours (NETs). Approximately 20% of tumours are still occult years later. Gallium-68 somatostatin receptor-PET/CT is promising for the detection of the causal primary NET, but its role in case of recurrent PCS is rarely reported. We report our experience with DOTATOC PET/CT in localising the causal NET in cases of initial but also recurrent PCS, and its clinical impact. A retrospective review of all DOTATOC PET/CTs performed in consecutive patients referred for PCS to our centre, between January 2011 and June 2017, was done. Nineteen patients underwent 26 PET/CTs, 13 for detection of a primary NET, seven for persistent or recurrent PCS after resection, and six for surveillance after resection of NETs previously detected on a DOTATOC PET/CT in our centre. Among the 13 PET/CTs performed to search for primary NET, five were positive: four carcinoid lung tumours were confirmed after resection and one lung focus was not confirmed since surgery would have carried a high risk. Clinical impact was 23% (3/13). Among the seven PET/CTs performed for persistent or recurrent PCS, six were true-positive, with confirmation of metastatic lymph nodes after resection. Clinical impact was 57% (4/7). All PET/CTs performed for surveillance were true-negative. DOTATOC PET/CT seems to be a valuable tool for detection of the NET responsible for persistent or recurrent PCS after surgery. In this context, DOTATOC PET/CT was more effective than for the detection of the causal tumour in initial PCS.

P2.03-10 Comprehensive Molecular Profiling and Comparison of Common and Rarer Subtypes of Lung Cancer

Genomic profiling of tumours has become a crucial component of precision cancer medicine. In order to comprehensively characterize molecular alterations in different lung cancer subtypes, we analysed a total number of 327 samples by using Whole-Exome Sequencing (WES) and SNP genotyping arrays. Additionally, we used Targeted Capture Sequencing (TCS) for scanning a selected panel of genes (n=52) at high sequencing depth. We WES (McGill University Innovation Centre in Montreal) 153 paired tumour-normal samples, with a further 174 paired tumour-normal samples undergoing TCS. Sequencing data were processed and mutations identified using BWA (v.0.7.15), Picard (v.2.17.11), GATK (v.3.7), VarScan (v.2.4.2) and VEP (v. 92) softwares. Illumina OmniExpressExome (v1.6) arrays were used for genotyping all samples and copy number alterations (CNAs) were identified using ASCAT (v.2.5.2), DNACopy (v.1.56.0) and GISTIC (v.2.0) softwares. The analysed samples had a tumour content varying from 20 to 90%. The age range of patients was between 28 to 89 years. Out of 159 lung cancer patients, 89 patients had lung adenocarcinoma (LUAD), 36 squamous cell carcinoma (LUSC) and 34 lung neuroendocrine (NET), of which 22 were subclassified as lung carcinoid (LC), 6 small cell carcinoma (LSCLC), 5 large cell carcinoma (LCNEC) and 1 combined NET subtype. TP53 appeared as the most mutated gene in LUSCs (82%), non-carcinoid NETs (58%) and LUADs (47%), but not in the LC subtype, while the chromatin remodelling gene ARID1A was altered across all subtypes (9%). Other mutated genes in LUAD were KRAS (31%), STK11 (22%), RBM10 (15%), EGFR (14%) and KEAP1 (14%); in LUSC were PTEN (26%), CDKN2A (21%), KEAP1 (21%) and NF1 (15%); in NET, non-carcinoid top mutated genes included RB1 (42%), ENPP2 (33%), ERBB4 and STK11 (17% for each), while ARID1A and ACKR3 were each present in 9.5 % of LC. In LUADs, mutations in EGFR and KRAS appeared as mutually-exclusive (P=0.007), while gene pairs NFE2L2 - AKT1 (P=0.012) and STK11- ALK (P=0.029) were co-mutated in LUAD and LUSC, respectively. Deletions in exons 19 and 20 of EGFR correlated with longer survival time compared to patients with wild-type EGFR (P=0.058). In NET, patients with mutated RB1 showed lower survival time compared to patients with wild-type RB1 (P=0.022). Examination of CNAs showed TERT amplifications (5p15.33 cytoband) were commonly found at high frequencies across all subtypes, especially in non-carcinoid NET (71.4%). Other recurrent CNAs included amplifications in MYC in 37% of LUAD and 40% of LUSC, and in EGFR in 33% of LUAD and 14% of LUSC. Deletions in the CDKN2A locus were seen at frequencies of 31% and 28% in LUAD and LUSC, respectively. LC patients showed longer survival time compared to other tumours (P=0.015). COSMIC mutational signatures 18 (of unknown aetiology) and 24 (associated with exposure to aflatoxin) were exclusively found in LC. The results confirm that lung cancer is a group of heterogenous diseases. In addition to the known effects of EFGR mutations, possible therapeutic avenues could be suggested for TERT amplifications, for which nucleoside analogues have shown to promote cancer cell death or EZH2 inhibitors for ARID1A-mutated cancers.

EP1.12-40 Neuroendocrine Tumors of the Lung: Clinico-Pathological Characterization and Follow-Up of Patients Treated at an Argentinean University Hospital in the Last 10 Years

Neuroendocrine tumors (NETs) comprise a heterogeneous group of malignancies that arise from neuroendocrine cells throughout the body, most commonly from the lungs and gastrointestinal tract. Histological characteristics and clinical behaviour define lung NETs, which are classified into four groups: typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine carcinoma (LCNC) and small-cell lung cancer (SCLC). The identification and differentiation of TCs from ACs or LCNECs and SCLC is essential for treatment options and prognosis. We performed a retrospective review of patients with lung NETs treated in our institution in the last decade. We analysed histological confirmed primary lung NETs cases. Demographics, clinical characteristics, imaging, treatment and outcome are described for this patient population. A total of 91 patients with lung NETs were included; 28/91 (30.8%) were lung carcinoids (TC:14 and AT: 14) and 63/91 (69.2%) were high grade lung NETs (SCLC: 54 and LCNC: 9). Comparing low and high grade NETs groups of patients, we could identify differences in the following variables: mean age at diagnosis (51±14 vs 61 ±9 years, p=0.000), mean time from the beginning of symptoms to achievement of pathologic diagnosis (10.5±15.9 vs 3.9 ± 2.6 months, p=0.002), ECOG³2 (14% vs 42%, p=0.001), smokers (57% vs 95%, p=0.000), median % of Ki67 expression (4%, (1-30%) vs 50%, (20-90), p=0.000), history of inflammatory lung disease (18% vs 32%, p=0.028) and presence extra-thoracic disease at diagnosis (47% vs 68%, p=0.000). No statistically differences between groups were observed in: sex, family history of lung cancer, second diagnosis of cancer and body mass index at diagnosis. Functioning carcinoid syndrome was observed in 10% (3/28) of patients with lung carcinoids. For the entire cohort, the median overall survival (OS) was 19 months (IC95%: 14-24 months). The five year OS for patients with lung carcinoids was 62% and 13% for high grade NETs. In our series of lung carcinoids we observed a high frequency of: atypical carcinoids, advanced disease at presentation and smoking history. However, the 5-year overall survival is the expected for this population. More knowledge is needed about this entity in South American countries.

ES04.05 Advances in Radionuclide Treatment for Neuroendocrine Tumours

Lung carcinoids (LC) are rare tumours, however incidence is increasing due to improvement in diagnostic techniques. They account for approximately 2% of all lung malignancies and around 20-30% of all neuroendocrine tumours (NETs). They characteristically have an indolent clinical behaviour with longer survival intervals compared to poorly differentiated lung neuroendocrine malignancies. LC are divided into typical or atypical carcinoid tumours according to pathological characteristics, such as amount of mitosis and necrosis. A significant proportion of LC expresses somatostatin receptors by immunohistochemistry. Nuclear medicine imaging, such as somatostatin receptor scintigraphy, has been employed for staging of LC for years. Development of new nuclear medicine imaging techniques, including Positron Emission Tomography (PET) combined with CT has improved diagnosis, staging and treatment of patients diagnosed with LC. 68-Gallium(68Ga)-radiolabelled PET (68Ga-DOTA-PET) tracers for functional NET imaging have emerged as potentially useful tools for diagnosis and staging. For localised stages of LCs, surgery is the treatment of choice, performed with curative intent. Locally advanced inoperable or metastatic tumours are treated with palliative approaches based on somatostatin analogues (SSAs), temozolomide-based chemotherapy combination and targeted therapies (everolimus). Recently, the use of Peptide Receptor Radionuclide Therapy (PRRT) has revolutionised the treatment of extra-pulmonary neuroendocrine tumours. Based on the results of the NETTER-1 study, PRRT has been approved for the management of small bowel and pancreatic NETs and it is considered a standard of care after progression to SSA for patients with uptake in 68Ga-DOTA-PET (theranostic approach). This lecture will summarise the state of the art of LC with a focus behind the rationale of PRRT and its potential role in the management of LCs. Neuroendocrine, PRRT, carcinoid

520P - Safety of high doses of somatostatin analogs in well differentiated NENs in elderly

BackgroundNeuroendocrine neoplasms (NENs) are a group of heterogeneous neoplasms which can be treated with different therapies: in well differentiated (WD) NENs analogs of somatostatin (SSA) can be used considering their proved antineoplastic and antisecretory effects. This is a multicenter study to investigate the safety of high doses (HDD) analogs of somatostatin (SSA) in WD NENs with particular attention for patients over 70 years of age. MethodsWe collected clinical data from 13 Italian NENs dedicated Units. Patients with WD NENs, in progression on previous treatments, included SSA, and subsequently treated with HDD-SSA, were considered. Results170 pts were included: 61.76% male, 38.24% female. The median age at the diagnosis was 58.83. Regarding the site of origin, the most frequent was gastro-intestinal system (54.71%), followed by pancreas (27.06%) and lung (8.24%). G1 NENs were 54.11%, G2 42.94% and typical lung carcinoid 2.95%; 31.76% were affected by a carcinoid syndrome or clinical conditions such as gastrinoma, insulinoma and Zollinger-Ellison. The median PFS was 20.18 months. Side effects (SEs) were registered in 25 pts (14.71%), the most frequent were cholecystitis (4%), G1 asthenia (12%), G1 hyperglycemia (8%), G1 steatorrhea (76%). We divided patients in 4 groups considering the age at the diagnosis: lower than 60 years, 61-70, 71-80 and more than 81 years. With the increase of age there was a higher percentage of SEs (Tab.1) with a maximum percentage of 20.41% in the group with an age of 71-80 years. The treatment was well tolerated without life-threatening side effects also in the group of patients with an age > 70 years. Tab. 1Table: 520PTable: 520PAge at the diagnosis (years)% side effects<6010,00%61-7013,73%71-8020,41%>8120,00% ConclusionsWe concluded that in this setting of patients, with an age >70 and a WD NEN in progression to standard doses of SSA, HD-SSA represents a safe and effective second line therapy with a median PFS of 20.80 months. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

P1.12-02 Nationwide Assessment of the Role of Adjuvant Systemic Therapy in High-Risk Lung Carcinoids

P1.12-02 Nationwide Assessment of the Role of Adjuvant Systemic Therapy in High-Risk Lung Carcinoids

Atypical diagnosis for typical lung carcinoid

BackgroundThe diagnosis of lung typical carcinoid tumors results challenging when limited size and unfavorable sampling location is associated. It has been reported that bronchoscopy with endobronchial ultrasound (EBUS) significantly increases the diagnostic yield of peripheral nodules smaller than 2 cm.Case presentationA 70-year-old Caucasian male complained of persistent fever and cough despite several antibiotic courses and steroid treatment. Chest radiology revealed the presence of a small single nodular opacity in the left upper lobe, whose standardized maximum uptake value (SUV) at fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET/CT) was significantly high (4.5). The patient underwent bronchial endoscopy but any appreciable sign of endobronchial or intramural involvement was detected. Only radial ultrasound-guided bronchoscopy (R-EBUS) allowed transbronchial sampling whose pathological analysis revealed a typical carcinoid tumor. The patients underwent surgical lobectomy and clinic-radiological follow was started.ConclusionsWith this case we aim at stressing the importance of ultrasound in the diagnostic process of lung small peripheral carcinoid, especially if they present without mucosal or sub mucosal involvement.

Integrative Genomic Characterization Identifies Molecular Subtypes of Lung Carcinoids.

Lung carcinoids (LC) are rare and slow growing primary lung neuroendocrine tumors. We performed targeted exome sequencing, mRNA sequencing, and DNA methylation array analysis on macro-dissected LCs. Recurrent mutations were enriched for genes involved in covalent histone modification/chromatin remodeling (34.5%; MEN1, ARID1A, KMT2C, and KMT2A) as well as DNA repair (17.2%) pathways. Unsupervised clustering and principle component analysis on gene expression and DNA methylation profiles showed three robust molecular subtypes (LC1, LC2, LC3) with distinct clinical features. MEN1 gene mutations were found to be exclusively enriched in the LC2 subtype. LC1 and LC3 subtypes were predominately found at peripheral and endobronchial lung, respectively. The LC3 subtype was diagnosed at a younger age than LC1 and LC2 subtypes. IHC staining of two biomarkers, ASCL1 and S100, sufficiently stratified the three subtypes. This molecular classification of LCs into three subtypes may facilitate understanding of their molecular mechanisms and improve diagnosis and clinical management. SIGNIFICANCE: Integrative genomic analysis of lung carcinoids identifies three novel molecular subtypes with distinct clinical features and provides insight into their distinctive molecular signatures of tumorigenesis, diagnosis, and prognosis.

Stage IV lung carcinoids: spectrum and evolution of proliferation rate, focusing on variants with elevated proliferation indices

The spectrum and evolution of proliferation rates in stage IV lung carcinoids is poorly defined. In particular, there are limited data on the prevalence and characteristics of tumors exceeding the standard upper proliferative criteria—as defined largely based on early-stage carcinoids—in metastatic setting. Sixty-six patients with stage IV lung carcinoids were identified, and all evaluable samples (n = 132; mean 2 samples per patient) were analyzed for mitotic counts and Ki-67 rate. Clinicopathologic and genomic features associated with elevated proliferation rates (>10 mitoses per 2 mm2 and/or >20% hot-spot Ki-67), and evolution of proliferation rates in serial specimens were analyzed. We found that mitoses and/or Ki-67 exceeded the standard criteria in 35 of 132 (27%) samples, primarily (31/35 cases) at metastatic sites. Although neuroendocrine neoplasms with >10 mitoses per 2 mm2 are currently regarded as de facto neuroendocrine carcinomas, the notion that these cases are part of the spectrum of carcinoids was supported by (1) well-differentiated morphology, (2) conventional proliferation rates in other samples from same patient, (3) genetic characteristics, including the lack of RB1/TP53 alterations in all tested samples (n = 19), and (4) median overall survival of 2.7 years, compared to <1 year survival of stage IV neuroendocrine carcinomas in the historic cohorts. In patients with matched primary/metastatic specimens (48 pairs), escalation of mitoses or Ki-67 by ≥10 points was observed in 35% of metastatic samples; clonal relationship in one pair with marked proliferative progression was confirmed by next-generation sequencing. Notably, escalation of proliferation rate was documented in a subset of metastases arising from resected typical carcinoids, emphasizing that the diagnosis of typical carcinoid in primary tumor does not assure low proliferation rate at metastatic sites. In conclusion, stage IV lung carcinoids frequently exceed the standard proliferative criteria established for primary tumors, and commonly exhibit proliferative escalation at metastatic sites. Despite the overlap of proliferation rates, these tumors show fundamental morphologic, genomic and clinical differences from neuroendocrine carcinomas, and should be classified separately from those tumors. Awareness of the increased proliferative spectrum in metastatic carcinoids is critical for their accurate diagnosis. Further studies are warranted to explore the impact of proliferation indices on prognosis and therapeutic responses of patients with metastatic carcinoids.

ACTH-producing tumorlets and carcinoids of the lung: clinico-pathologic study of 63 cases and review of the literature.

Adrenocorticotropic hormone (ACTH)-secreting lung carcinoids represent the principal cause of ectopic Cushing syndrome, but the prevalence of ACTH expression and the association between ACTH production and Cushing syndrome in lung carcinoids have scarcely been investigated. In addition, available information on the prognostic meaning of ACTH production is controversial. The aims of this multicentric retrospective study, also including a review of the literature, were to describe the clinico-pathologic features of ACTH-producing lung carcinoids, to assess recurrence and specific survival rates, and to evaluate potential prognostic factors. To identify ACTH production in 254 unselected and radically resected lung carcinoids, we used a double approach including RT-PCR (mRNA encoding for pro-opiomelanocortin) and immunohistochemistry (antibodies against ACTH and β-endorphin). Sixty-three (24.8%) tumors produced ACTH and 11 of them (17.4%), representing 4.3% of the whole series, were associated with Cushing syndrome. The median follow-up time was 71months. The 10-year overall and specific survival rates were 88.5% and 98.2%, respectively, with difference neither between functioning and nonfunctioning tumors nor between ACTH-positive and ACTH-negative carcinoids. At univariate analysis, histological type (typical or atypical) and Ki67 index significantly correlated with tumor recurrence. The literature review identified 172 previously reported patients with functioning ACTH-secreting lung carcinoids, and the meta-analysis of survival showed that 92% of them were alive after a mean follow-up time of 50months. Our results demonstrate that ACTH-producing lung carcinoids are not rare, are not always associated with Cushing syndrome, and do not represent an aggressive variant of lung carcinoid.

Prognostic value of cyclin A2 and B1 expression in lung carcinoids

Carcinoid classification in the lung is still based on morphological criteria. Although there are many studies investigating the role of Ki-67 proliferation index in the classification of lung neuroendocrine tumours, it is still not used in routine diagnostics. Interestingly, cyclins, which have a crucial role in controlling the cell cycle, have not been thoroughly studied in lung neuroendocrine tumours. The aim of our study was to investigate the correlation of cyclin A2 and B1 expression with prognosis, Ki-67 proliferation index, and carcinoid morphology. A cohort of 134 resected typical and atypical carcinoids was stained with antibodies against Ki-67, cyclin A2 and B1. The positive nuclear reaction was assessed in hot spot areas and expressed as the percentage of tumour cells. Univariate analyses found the highest relative hazard between low and high cyclin A2 expression both with respect to overall survival [hazard ratio (HR)=16; 95% confidence interval (CI) 4.8-51; p=0.0000054], and relapse (HR=8; 95% CI 3.1-21; p=0.00002). In multivariate analysis for overall survival cyclin A2 (HR=10; 95% CI 2.5->100; p=0.0082) and B1 (HR=6.5; 95% CI 1.5-35; p=0.02) remained significant when adjusted for other risk factors, whereas Ki-67 was no longer significant (HR=0.64; 95% CI 0.003-5.5; p=0.65). This suggests that Ki-67 is closer to conventional risk factors for survival than cyclin A2 and B1. Furthermore, the analysis revealed 4 mitoses per 2 mm2 as a more powerful prognostic cut-off than currently accepted 2 mitoses. We have clearly demonstrated that application of cyclin A2 and cyclin B1 might bring additional value regarding the overall and progression-free survival of patients with carcinoids of the lung.

Multiple Assays to Determine Methylguanine-Methyltransferase Status in Lung Carcinoids and Correlation with Clinical and Pathological Features

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.

Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

Background. The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. KEAP1 silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer. Methods. We performed a detailed methylation density map of 13 CpGs located into the KEAP1 promoter region by analyzing a set of 25 cell lines from different histologies of lung cancer. The methylation status was assessed using quantitative methylation specific PCR (QMSP) and pyrosequencing, and the performance of the two assays was compared. Results. Hypermethylation at the promoter region of the KEAP1 was detected in one third of cell lines and its effect on the modulation KEAP1 mRNA levels was also confirmed by in vitro 5-Azacytidine treatment on lung carcinoid, small lung cancer and adenocarcinoma cell lines. QMSP and pyrosequencing showed a high rate of concordant results, even if pyrosequencing revealed two different promoter CpGs sub-islands (P1a and P1b) with a different methylation density pattern. Conclusions. Our results confirm the effect of methylation on KEAP1 transcription control across multiple histologies of lung cancer and suggest pyrosequencing as the best approach to investigate the pattern of CpGs methylation in the promoter region of KEAP1. The validation of this approach on lung cancer patient cohorts is mandatory to clarify the prognostic value of the epigenetic deregulation of KEAP1 in lung tumors.