A neuroligin-2-YAP axis regulates progression of pancreatic intraepithelial neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a dismal prognosis that arises from precursor lesions called pancreatic intraepithelial neoplasias (PanINs). Progression from low- to high-grade PanINs is considered as tumor initiation, and a deeper understanding of this switch is needed. Here, we show that synaptic molecule neuroligin-2 (NLGN2) is expressed by pancreatic exocrine cells and plays a crucial role in the regulation of contact inhibition and epithelial polarity, which characterize the switch from low- to high-grade PanIN. NLGN2 localizes to tight junctions in acinar cells, is diffusely distributed in the cytosol in low-grade PanINs and is lost in high-grade PanINs and in a high percentage of advanced PDACs. Mechanistically, NLGN2 is necessary for the formation of the PALS1/PATJ complex, which in turn induces contact inhibition by reducing YAP function. Our results provide novel insights into NLGN2 functions outside the nervous system and can be used to model PanIN progression.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Impact of Caloric Restriction in Breast Cancer Patients Treated with Neoadjuvant Chemotherapy: A Prospective Case Control Study.

Background and aims: It is well established that caloric restriction (CR) may influence metabolic and hormonal factors involved in cancer development and progression. Recently, several studies have demonstrated that CR may have a favorable impact on the response to systemic therapy in breast cancer (BC) patients. However, there is a lack of data regarding the influence of CR during neoadjuvant chemotherapy (NACT). Our study's primary aim was to evaluate CR's impact on BC patients undergoing NACT. Secondly, we investigated the nutritional efficacy and safety of this intervention. Methods: We performed a prospective, case-control study in two breast units. A diet group consisting of 39 patients undergoing NACT and CR was enrolled in our study at the same time. CR consisted of a 30% reduction in caloric intake, which increased to 50% on the days before, during, and after the administration of chemotherapy. A control group of 60 patients that underwent the same treatment approach only followed the general dietary recommendations for BC according to WCRF guidelines. The diet group was monitored during the study for both dietary adequacy and weight trends. Results: CR combined with NACT showed a statistically significant therapeutic response in tumor size (OR 2.94, IC 1.07-8.01, p = 0.009) and lymph node status (OR 3.22, IC 1.22-8.56, p = 0.001) compared to NACT alone, even after the adjustment for all biological parameters. Our data also showed the efficacy and safety of this intervention in both anthropometric and biochemical analyses. Conclusions: Patients who adhered to CR showed a better response to NACT, both in the breast and in the axillary lymph nodes, compared to the patients in the control group. Furthermore, the CR diet combined with NACT showed good tolerance and safety.

Clinical and Pathological Features and Gene Expression Profiles of Clinically Aggressive Papillary Thyroid Carcinomas.

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.

Neuroendocrine neoplasms of the breast: diagnostic agreement and impact on outcome.

The classification of breast neuroendocrine neoplasms (Br-NENs) was modified many times over the years and is still a matter of discussion. In the present study, we aimed to evaluate the diagnostic reproducibility and impact on patient outcomes of the most recent WHO 2019 edition of breast tumor classification, namely, for neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs). This multicentric observational study included 287 breast neoplasms with NE differentiation. The cases were blindly classified by three independent groups of dedicated breast and/or endocrine pathologists following the 2019 guidelines. Diagnostic concordance and clinical impact were assessed. We observed only a moderate overall diagnostic agreement across the three centers (Cohen's kappa 0.4532) in distinguishing NET from solid papillary carcinomas (SPCs) and no special type carcinomas (NST) with NE differentiation. Br-NENs were diagnosed in 122/287 (42.5%) cases, subclassified as 11 NET G1 (3.8%), 84 NET G2 (29.3%), and 27 NEC (9.4%), the latter group consisting of 26 large-cell and 1 small-cell NECs. The remaining 165/287 (57.5%) cases were labeled as non-NEN, including SPC, mucinous, NST, and mixed NE carcinomas. While NET and non-NEN cases had a comparable outcome, the diagnosis of NECs showed negative impact on disease-free interval compared to NETs and non-NENs (p = 0.0109). In conclusion, the current diagnostic classification of Br-NENs needs further adjustments regarding morphological and immunohistochemical criteria to increase the diagnostic reproducibility among pathologists. Our data suggest that, apart from high-grade small- and large-cell NECs, Br-NENs behave like non-NEN breast carcinomas and should be managed similarly.

Stain normalization in digital pathology: Clinical multi-center evaluation of image quality.

In digital pathology, the final appearance of digitized images is affected by several factors, resulting in stain color and intensity variation. Stain normalization is an innovative solution to overcome stain variability. However, the validation of color normalization tools has been assessed only from a quantitative perspective, through the computation of similarity metrics between the original and normalized images. To the best of our knowledge, no works investigate the impact of normalization on the pathologist's evaluation. The objective of this paper is to propose a multi-tissue (i.e., breast, colon, liver, lung, and prostate) and multi-center qualitative analysis of a stain normalization tool with the involvement of pathologists with different years of experience. Two qualitative studies were carried out for this purpose: (i) a first study focused on the analysis of the perceived image quality and absence of significant image artifacts after the normalization process; (ii) a second study focused on the clinical score of the normalized image with respect to the original one. The results of the first study prove the high quality of the normalized image with a low impact artifact generation, while the second study demonstrates the superiority of the normalized image with respect to the original one in clinical practice. The normalization process can help both to reduce variability due to tissue staining procedures and facilitate the pathologist in the histological examination. The experimental results obtained in this work are encouraging and can justify the use of a stain normalization tool in clinical routine.

FOXA1 in Breast Cancer: A Luminal Marker with Promising Prognostic and Predictive Impact.

The present review focuses on the function of the forkhead protein FOXA1 in breast cancer (BC) in relation to steroid hormone receptors. We explored the currently available analytic approaches for FOXA1 assessment both at gene and protein levels, comparing the differences between the available techniques used for its diagnostic assessment. In addition, we elaborated on data regarding the prognostic and predictive role of this marker in BC based on several studies that evaluated its expression in relation to the outcome and/or response to therapy. FOXA1, similar to the androgen receptor (AR), may have a dual role in BC according to hormonal status. In luminal cancers, its expression contributes to a better prognosis, while in triple-negative breast cancers (TNBC), it implies an adverse outcome. Consequently, we observed that FOXA1-positive expression in a neoadjuvant setting may predict a lack of response in luminal BC as opposed to TNBC, in which FOXA1 allegedly increases its chemosensitivity. In conclusion, considering its accessible and convenient identification by immunohistochemistry, its important impact on prognosis, and its suitability to identify patients with different responses to chemotherapy, we propose that FOXA1 could be tested in routine diagnostics as an additional prognostic and predictive marker in BC.

Overexpression of INSM1, NOTCH1, NEUROD1, and YAP1 genes is associated with adverse clinical outcome in pediatric neuroblastoma.

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.

Molecular Subtypes of Extra-pulmonary Neuroendocrine Carcinomas Identified by the Expression of Neuroendocrine Lineage-Specific Transcription Factors.

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.

Automated Analysis of Proliferating Cells Spatial Organisation Predicts Prognosis in Lung Neuroendocrine Neoplasms.

Lung neuroendocrine neoplasms (lung NENs) are categorised by morphology, defining a classification sometimes unable to reflect ultimate clinical outcome. Subjectivity and poor reproducibility characterise diagnosis and prognosis assessment of all NENs. Here, we propose a machine learning framework for tumour prognosis assessment based on a quantitative, automated and repeatable evaluation of the spatial distribution of cells immunohistochemically positive for the proliferation marker Ki-67, performed on the entire extent of high-resolution whole slide images. Combining features from the fields of graph theory, fractality analysis, stochastic geometry and information theory, we describe the topology of replicating cells and predict prognosis in a histology-independent way. We demonstrate how our approach outperforms the well-recognised prognostic role of Ki-67 Labelling Index on a multi-centre dataset comprising the most controversial lung NENs. Moreover, we show that our system identifies arrangement patterns in the cells positive for Ki-67 that appear independently of tumour subtyping. Strikingly, the subset of these features whose presence is also independent of the value of the Labelling Index and the density of Ki-67-positive cells prove to be especially relevant in discerning prognostic classes. These findings disclose a possible path for the future of grading and classification of NENs.

Caveolin-1 in Kidney Chronic Antibody-Mediated Rejection: An Integrated Immunohistochemical and Transcriptomic Analysis Based on the Banff Human Organ Transplant (B-HOT) Gene Panel.

Caveolin-1 overexpression has previously been reported as a marker of endothelial injury in kidney chronic antibody-mediated rejection (c-ABMR), but conclusive evidence supporting its use for daily diagnostic practice is missing. This study aims to evaluate if Caveolin-1 can be considered an immunohistochemical surrogate marker of c-ABMR. Caveolin-1 expression was analyzed in a selected series of 22 c-ABMR samples and 11 controls. Caveolin-1 immunohistochemistry proved positive in peritubular and glomerular capillaries of c-ABMR specimens, irrespective of C4d status whereas all controls were negative. Multiplex gene expression profiling in c-ABMR cases confirmed Caveolin-1 overexpression and identified additional genes (n = 220) and pathways, including MHC Class II antigen presentation and Type II interferon signaling. No differences in terms of gene expression (including Caveolin-1 gene) were observed according to C4d status. Conversely, immune cell signatures showed a NK-cell prevalence in C4d-negative samples compared with a B-cell predominance in C4d-positive cases, a finding confirmed by immunohistochemical assessment. Finally, differentially expressed genes were observed between c-ABMR and controls in pathways associated with Caveolin-1 functions (angiogenesis, cell metabolism and cell-ECM interaction). Based on our findings, Caveolin-1 resulted as a key player in c-ABMR, supporting its role as a marker of this condition irrespective of C4d status.

Adrenal Rests in the Uro-genital Tract of an Adult Population.

Ectopic adrenal rests are a rare condition which can be found in various sites, generally in the retroperitoneum or pelvis along the path of gonadal descent. Their real prevalence is unknown. Males are more commonly affected, at least in the pediatric age. Adrenal rests are usually clinically silent and incidentally found in surgical samples, mostly in the pediatric population, and rarely in adults. With the aim of increasing knowledge and estimating the prevalence of ectopic adrenocortical tissue in the adult population, 44 adrenal rests in the urogenital tract of 40 adults are described. These represent approximately 0.07% of the total number of urogenital and gynecological surgeries performed in the 22 considered years. Adrenal rests were identified in the spermatic cord (10 males) and in paraovarian, parasalpingeal, or infundibulopelvic ligament locations (30 females). All but one was incidental findings. One case regarded an adrenocortical carcinoma arisen in adrenal rests. A literature review of adrenal ectopia in the urogenital tract of adults identified 57 reported cases from 53 patients, with similar clinicopathological features as those of our series, with the exception of a lower incidence of parasalpingeal locations. Despite their limited clinical implications, awareness of ectopic adrenal rests is essential also in adults for at least two reasons: (a) to correctly identify sources of adrenocortical hormone production in case of adrenal insufficiency or hormonal imbalance and (b) to avoid misinterpretations in the diagnostic workup of renal cell carcinoma, adrenocortical tumors, and rare gonadal neoplasms, including Sertoli/Leydig cell tumors.

Differential Expression Profiles of Cell-to-Matrix-Related Molecules in Adrenal Cortical Tumors: Diagnostic and Prognostic Implications.

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.

INSM1 Expression in Breast Neoplasms with Neuroedocrine Features.

According to the 2019 WHO classification of breast tumors, neuroendocrine neoplasms (NENs) are classified into well-differentiated NE tumors (NET) and poorly differentiated NE carcinomas (NEC), while other breast cancers (BCs) of special and no special type with neuroendocrine (NE) features are not incorporated in this scheme anymore. We aimed to assess whether INSM1, a novel NE marker, could have a role in breast NEN subtyping. We selected 63 BCs operated from 2003 to 2018, classified as BCs with NE features, with available clinico-pathological data. Following 2019 WHO criteria, this cohort was reclassified into 37 NETs/NECs, the remaining 26 tumors representing solid-papillary (7), mucinous (7), and mixed type (12) carcinomas with NE differentiation. Chromogranin A (CGA) and synaptophysin (SYN) immunostains were reviewed, and INSM1 was tested by immunohistochemistry. Thirty CGA- and SYN-negative no special type BCs served as negative control. INSM1 was expressed in 52/63 cases of the whole cohort (82.54%). INSM1 positive and negative cases had no significantly different clinico-pathological characteristics. INSM1 expression was not significantly different between the newly reclassified NET/NEC group and other BCs with NE features. No immunoexpression was observed in control BCs. The sensitivity and specificity of INSM1 for the NE phenotype was 82.5% and 100%, respectively, compared to 61.9% and 100% for CGA, and 95.2 and 100% for SYN. In conclusion, INSM1 is as accurate as traditional NE biomarkers to identify NE differentiation in BC. In analogy to standard NE markers, INSM1 could not distinguish NET and NEC from the other BC histotypes with NE differentiation.

Clinical Relevance of Tubular Breast Carcinoma: Large Retrospective Study and Meta-Analysis.

Background: Tubular carcinoma (TC) is a low proliferative grade 1 (G1) breast cancer (BC). Despite its favorable outcome and allegedly lower aggressiveness, patients are treated like other luminal G1 BC, with radiotherapy (RT) and hormonal therapy (HT). We performed: (1) a retrospective study comparing a TC cohort and a control series of luminal G1 BC and (2) a systematic review and meta-analysis focused on TC outcome. Materials and Methods: We selected a series of 572 G1 luminal BC patients [111 TC, 350 not otherwise specified (NOS), and 111 special-type (ST) BC] with follow-up and clinico-pathological data, who underwent local excision followed by RT at Città della Salute e della Scienza Hospital, Turin. Moreover, 22 and 13 studies were included in qualitative and quantitative meta-analysis, respectively. Results: TCs were generally smaller (≤10 mm) (P < 0.001), with lower lymph node involvement (P < 0.001). TCs showed no local and/or distant recurrences, while 16 NOS and 2 ST relapsed (P = 0.036). Kaplan-Meier curves confirmed more favorable TC outcome (DFI: log-rank test P = 0.03). Meta-analysis data, including the results of our study, showed that the pooled DFI rate was 96.4 and 91.8% at 5 and 10 years, respectively. Meta-regression analyses did not show a significant influence of RT nor HT on the DFI at 10 years. Conclusions: Compared to the other G1 BCs, TCs have an excellent outcome. The meta-analysis shows that TC recurrences are infrequent, and HT and RT have limited influence on prognosis. Hence, accurate diagnosis of TC subtype is critical to ensuring a tailored treatment approach.

Diagnostic Value of Conventional PET Parameters and Radiomic Features Extracted from 18F-FDG-PET/CT for Histologic Subtype Classification and Characterization of Lung Neuroendocrine Neoplasms.

AIM: To evaluate if conventional Positron emission tomography (PET) parameters and radiomic features (RFs) extracted by 18F-FDG-PET/CT can differentiate among different histological subtypes of lung neuroendocrine neoplasms (Lu-NENs). METHODS: Forty-four naïve-treatment patients on whom 18F-FDG-PET/CT was performed for histologically confirmed Lu-NEN (n = 46) were retrospectively included. Manual segmentation was performed by two operators allowing for extraction of four conventional PET parameters (SUVmax, SUVmean, metabolic tumor volume (MTV), and total lesion glycolysis (TLG)) and 41 RFs. Lu-NENs were classified into two groups: lung neuroendocrine tumors (Lu-NETs) vs. lung neuroendocrine carcinomas (Lu-NECs). Lu-NETs were classified according to histological subtypes (typical (TC)/atypical carcinoid (AC)), Ki67-level, and TNM staging. The least absolute shrink age and selection operator (LASSO) method was used to select the most predictive RFs for classification and Pearson correlation analysis was performed between conventional PET parameters and selected RFs. RESULTS: PET parameters, in particular, SUVmax (area under the curve (AUC) = 0.91; cut-off = 5.16) were higher in Lu-NECs vs. Lu-NETs (p < 0.001). Among RFs, HISTO_Entropy_log10 was the most predictive (AUC = 0.90), but correlated with SUVmax/SUVmean (r = 0.95/r = 0.94, respectively). No statistical differences were found between conventional PET parameters and RFs (p > 0.05) and TC vs. AC classification. Conventional PET parameters were correlated with N+ status in Lu-NETs. CONCLUSION: In our study, conventional PET parameters were able to distinguish Lu-NECs from Lu-NETs, but not TC from AC. RFs did not provide additional information.

Recent advances and current controversies in lung neuroendocrine neoplasms✰.

In the lung, neuroendocrine tumors (NETs), namely typical and atypical carcinoids, and neuroendocrine carcinomas (NECs), grouping small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), make up for distinct tumor entities according to epidemiological, genetic, pathologic and clinical data. The proper classification is essential in clinical practice for diagnosis, prognosis and therapy purposes. Through an extensive literature survey, three perspectives on lung NENs have been revised: i) criteria and terminology on biopsy or cytology samples of primaries or metastases; ii) carcinoids with elevated mitotic counts and/or Ki-67 proliferation rates; iii) relevance of molecular landscape to identify new tumor entities and therapeutic targets. Furthermore, a dispute about lung NEN development has been raised according to emerging molecular models. We herein provide a pathology update on practical topics in the setting of lung NENs according to the current classification (recent advances). We have also reappraised the development of these tumors by modeling risk factors and natural history of disease (recent controversies). Combining recent advances and controversies may help clarify our biological understanding of lung NENs and give practical information for the clinical decision-making process.