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The introduction of Ki67 immunohistochemistry in the work-up of neuroendocrine neoplasms (NENs) has opened a new approach for their diagnosis and prognostic evaluation. Since the first demonstration of the prognostic role of Ki67 proliferative index in pancreatic NENs in 1996, several studies have been performed to explore its prognostic, diagnostic, and predictive role in other neuroendocrine and endocrine neoplasms. A large amount of information is now available and published results globally indicate that Ki67 proliferative index is useful to this scope, although some differences exist in relation to tumor site and type. In gut and pancreatic NENs, the Ki67 proliferative index has a well-documented and accepted diagnostic and prognostic role and its evaluation is mandatory in their diagnostic work-up. In the lung, the Ki67 index is recommended for the diagnosis of NENs on biopsy specimens, but its diagnostic role in surgical specimens still remains to be officially accepted, although its prognostic role is now well documented. In other organs, such as the pituitary, parathyroid, thyroid (follicular cell-derived neoplasms), and adrenal medulla, the Ki67 index does not play a diagnostic role and its prognostic value still remains a controversial issue. In medullary thyroid carcinoma, the Ki67 labelling index is used to define the tumor grade together with other morphological parameters, while in the adrenal cortical carcinoma, it is useful to select patients to treated with mitotane therapy. In the present review, the most important information on the diagnostic, prognostic, and predictive role of Ki67 proliferative index is presented discussing the current knowledge. In addition, technical issues related to the evaluation of Ki67 proliferative index and the future perspectives of the application of Ki67 immunostaining in endocrine and neuroendocrine neoplasms is discussed.

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Preclinical data show that intravesical instillation of Ty21a/Vivotif, a commercial vaccine against typhoid fever, is an effective alternative option to standard Bacillus Calmette–Guérin (BCG) immunotherapy for non–muscle-invasive bladder cancer (NMIBC). Here, we characterized the inflammatory effects of Ty21a on the bladder and investigated the immune mechanisms underlying tumor regression toward the use of this bacterial vaccine in NMIBC patients. MB49 bladder tumor–bearing mice had significantly improved survival after intravesical instillations of Ty21a doses of 106 to 108 colony-forming units. By IHC and morphology, both BCG and Ty21a instillations were associated with bladder inflammation, which was decreased with the use of low, but effective doses of Ty21a. Flow-cytometry analysis showed a significant infiltration of T cells, natural killer (NK) cells, and myeloid cells, compared with controls, after a single dose of Ty21a, whereas this was only observed after multiple doses of BCG. The induced myeloid cells were predominantly neutrophils and Ly6C+CD103+ dendritic cells (DC), the latter being significantly more numerous after instillation of Ty21a than BCG. Ex vivo infection of human leukocytes with Ty21a, but not BCG, similarly significantly increased DC frequency. CD4+ and CD8+ T cells, but not NK cells nor neutrophils, were required for effective bladder tumor regression upon Ty21a treatment. Thus, the generation of antitumor adaptive immunity was identified as a key process underlying Ty21a-mediated treatment efficacy. Altogether, these results demonstrate mechanisms behind intravesical Ty21a therapy and suggest its potential as a safe and effective treatment for NMIBC patients.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Little is known as to whether there may be any pathogenetic link between pulmonary carcinoids and neuroendocrine carcinomas. An eight-gene signature with copy number variations (CNVs) in neuroendocrine neoplasms (NENs), namely MEN1, MYC, MYCL1, RICTOR, RB1, SDHA, SRC and TP53, was used to classify an independent cohort of 54 surgically resectable tumors [31 typical carcinoids (TC), 11 atypical carcinoids (AC) and 12 small cell lung carcinomas (SCLC)], for which transcriptome and mutation data were available. Unsupervised clustering analysis identified two histology-independent clusters, namely CL1 and CL2, where 17/42 (40.5%) carcinoids and all the SCLC samples fell into the latter. CL2 carcinoids affected survival adversely, were enriched in T to G transversions and T>C/C>T transitions in the context of specific mutational signatures, presented with at least 1.5-fold change (FC) increase of gene mutations including TSC2, SMARCA2, SMARCA4, ERBB4 and PTPRZ1, differed for gene expression and showed epigenetic changes in charge of MYC and MTORC1 pathways, cellular senescence, inflammation, high- plasticity cell state and immune system exhaustion. Similar results were also confirmed in an independent validation set of 101 lung NENs (24 carcinoids, 21 SCLC and 56 LCNEC). We herein propose an unexpected shifting to the current pathogenesis paradigm, suggesting that some NECs could secondarily originate from a subset of genomically transformed carcinoids prone to epigenetic mechanism-driven progression.

Extra-pituitary ACTH secretion is associated with a variety of neoplastic conditions and may cause the so-called ectopic ACTH-dependent Cushing syndrome (CS). The clarification of the mechanisms of extra-pituitary ACTH expression would provide potential therapeutic targets for this complex and severe disease. In the adenohypophysis, the transcription factor TPIT, co-operating with other molecules, induces POMC expression and ACTH production. However, no data is currently available on the presence and role of TPIT expression in extra-pituitary ACTH-producing neoplasms. This study was designed to explore TPIT expression in a series of pulmonary and pancreatic ACTH-producing tumors, either CS-associated or not. 41 extra-pituitary ACTH-producing neuroendocrine tumors (NETs) were included in the study, encompassing 32 NETs of the lung (LuNETs), 7 of the pancreas (PanNETs) and 2 pheochromocytomas. Of these, 9 LuNETs, all PanNETs and the two pheochromocytomas were CS-associated. For comparison, 6 NETs the pituitary gland (PitNETs), and 35 ACTH-negative NETs (15 Lu-NETs and 20 PanNETs) were analyzed. Immunohistochemistry with specific anti-TPIT antibodies and quantitative real time PCR (qRT-PCR) were performed using standard protocols. TPIT expression was completely absent (protein and mRNA) in PanNETs, pheochromocytomas, and all ACTH-negative NETs. In contrast, it was expressed in 16/32 LuNETs, although with lower levels than in PitNETs. No definite relationship was found between immunohistochemistry TPIT expression and NET grade or the presence of Cushing syndrome. This study further highlights the clinical and biological heterogeneity of extrapituitary ACTH secretion and suggests that the differences between ACTH-secreting PanNETs and LuNETs may mirror distinct molecular mechanisms underlying POMC expression. Our results point towards the recognition of a real corticotroph-like phenotype of ACTH-producing LuNETs, that is not a feature of ACTH-producing PanNETs.

Criteria for the interpretation of digestive system neuroendocrine neoplasms (NENs) continue to evolve. Although there are some literature recommendations regarding workup and diagnosis of these lesions, different practice patterns exist among pathologists when signing out these specimens. The aim of this study was to assess practice trends among pathologists worldwide when reporting these neoplasms.We created an online survey with multiple questions pertaining to digestive NENs. The results were analysed based on type of practice setting, years of sign-out experience, and practice location. Respondents included 384 practicing pathologists: 70% academic, 30% private practice; 63% gastrointestinal (GI) pathology-subspecialised, 37% not; 39% North American, 42% European, 19% others; 45% with ≤10 years in practice; 55% with10 years. Some question responses were chosen by the majority (e.g. 85% use both mitotic count and Ki67 index for grading NENs, 82% complete a synoptic, and Ki67 stain even for small incidental appendiceal neuroendocrine tumours [NETs], and 96% utilize the diagnosis of grade 3 NET). However, some questions showed varying responses, including counting mitotic figures, Ki67 stain interpretation, and pancreatic grade 3 NEN workup. Pathologists also had some variability in interpreting regional metastatic foci of small bowel NETs and in choosing blocks for Ki67 staining in multifocal lesions.There existed scenarios wherein practice patterns varied despite recommendations in the literature, and there were also scenarios lacking clear guidelines wherein pathologists used varying judgement. This survey highlights current key grey areas in digestive system NEN evaluation, leading to variation in practice patterns.

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.

Neuroendocrine neoplasms (NENs) of the major and minor ampulla are rare diseases with clinico-pathologic features distinct from non-ampullary-duodenal NENs. However, they have been often combined and the knowledge on prognostic factors specific to ampullary NENs (Amp-NENs) is limited. The aim of this study was to identify factors associated with metastatic potential and patient prognosis in Amp-NENs. We clinically and histologically investigated an international series of 119 Amp-NENs, comprising 93 ampullary neuroendocrine tumors (Amp-NETs) and 26 neuroendocrine carcinomas (Amp-NECs). Somatostatin-producing tubulo-acinar NET represented the predominant Amp-NET histologic subtype (58 cases, 62%, 12 associated with type 1 neurofibromatosis). Compared to Amp-NETs, Amp-NECs arose in significantly older patients and showed a larger tumor size, a more frequent small vessel invasion, a deeper level of invasion and a higher rate of distant metastasis, and, importantly, a tremendously worse disease-specific patient survival. In Amp-NETs, the WHO grade proved to be a strong predictor of disease-specific survival (hazard ratio: 12.61, p 60 years, small vessel invasion, pancreatic invasion, and distant metastasis at diagnosis. Although nodal metastatic disease was not associated with survival by itself, patients with > 3 metastatic lymph nodes showed a worse outcome in comparison with the remaining Amp-NET cases with lymphadenectomy. Tumor epicenter in the major ampulla, small vessel invasion, and tumor size > 16 mm were independent predictors of nodal metastases in Amp-NETs. In conclusion, we identified prognostic factors, which may eventually help guide treatment decisions in Amp-NENs.

Neuroendocrine neoplasms (NENs) are a heterogeneous group of neoplastic proliferations showing different morphological features, immunophenotype, molecular background, clinical presentation, and outcome. They can virtually originate in every organ of the human body and their classification is not uniform among different sites. Indeed, as they have historically been classified according to the organ in which they primarily arise, the different nomenclature that has resulted have created some confusion among pathologists and clinicians. Although a uniform terminology to classify neuroendocrine neoplasms arising in different systems has recently been proposed by WHO/IARC, some issues remain unsolved or need to be clarified. In this review, we discuss the lights and shadows of the current WHO classifications used to define and characterize NENs of the pituitary gland, lung, breast and those of the head and neck region, and digestive and urogenital systems.

Sinonasal neuroendocrine neoplasms (SN-NENs) are rare and mostly include neuroendocrine carcinoma (NEC), whereas neuroendocrine tumor (NET) is exceptional in this site. Olfactory neuroblastoma (ONB) is a malignant neuroectodermal neoplasm arising in the nasal cavity. Albeit crucial for correct patients’ management, the distinction of high grade ONB from NEC is challenging and requires additional diagnostic markers. The transcription factor SATB2 has been recently introduced in routine diagnostics as an immunohistochemical marker of distal intestine differentiation. No specific data are available about SATB2 and GATA3 expression in SN-NENs. GATA3, SATB2, and, for comparison, CDX2 expression were investigated in a series of epithelial and non-epithelial SN-NENs. We collected 26 cases of ONB and 7 cases of epithelial SN-NENs diagnosed and treated in our Institution. ONBs were graded according to Hyams’ system and epithelial NENs were reclassified into 5 NECs, 1 MiNEN, and 1 amphicrine carcinoma. Immunohistochemistry was performed using standard automated protocols. Hyams’ grades 1–3 ONBs stained diffusely and intensely for SATB2, whereas grade 4 ONBs and NECs were globally negative. The non-neuroendocrine component of MiNEN and the amphicrine carcinoma were strongly positive. GATA3 was heterogeneously and unpredictably expressed in Hyams’ grades 1–3 ONBs, whereas grade 4 ONBs and NECs were completely negative. CDX2 was negative in all cases. Our study identifies, for the first time, SATB2 and GATA3 expression as features of Hyams’ grades 1–3 ONBs, expands the spectrum of SATB2 and GATA3-positive neoplasms, and suggests that Hyams’ grade 4 ONBs are not only clinically but also biologically different from low graded ONBs.

Primary non-neuroendocrine tumours of the pituitary gland and sella are rare lesions often challenging to diagnose. We describe two cases of clinically aggressive primary glomus tumour of the pituitary gland. The lesions occurred in a 63-year-old male and a 30-year-old female who presented with headache, blurred vision and hypopituitarism. Neuroimaging demonstrated large sellar and suprasellar tumours invading the surrounding structures. Histologically, the lesions were characterised by angiocentric sheets and nests of atypical cells that expressed vimentin, smooth muscle actin and CD34. Perivascular deposition of collagen IV was also a feature. Case 2 expressed synaptophysin. INI-1 (SMARCB1) expression was preserved. Both lesions were mitotically active and demonstrated a Ki-67 labelling index of 30%. Next-generation sequencing performed in case 1 showed no mutations in the reading frame of 37 commonly mutated oncogenes, including BRAF and KRAS. Four pituitary glomus tumours have previously been reported, none of which showed features of malignant glomus tumour. Similar to our two patients, three previous examples displayed aggressive behaviour.

The exact prediction of outcome of patients with Merkel cell carcinoma (MCC) of the skin is difficult to determine, although several attempts have been made to identify clinico-pathologic prognostic factors. The Ki67 proliferative index is a well-known marker routinely used to define the prognosis of patients with neuroendocrine neoplasms. However, its prognostic value has been poorly investigated in MCC, and available published results are often contradictory mainly because restricted to small series in the absence of standardized methods for Ki67 evaluation. For this reason, we explored the potential prognostic role of Ki67 proliferative index in a large series of MCCs using the WHO standardized method of counting positive cells in at least 500 tumor cells in hot spot areas on camera-captured printed images. In addition, since MCC may be considered as the cutaneous counterpart of digestive neuroendocrine carcinomas (NECs), we decided to stratify MCCs using the available and efficient Ki67 threshold of 55%, which was found prognostic in digestive NECs. This choice was also supported by the Youden index analysis. In addition, we analyzed the prognostic value of other clinico-pathologic parameters using both univariate and multivariate analysis. Ki67 index appeared significantly associated with prognosis at univariate analysis together with stage IV, lack of MCPyV, and p63 expression, but not at the multivariate analysis, where survival resulted independently influenced by p63 expression and tumor stage, only.

Neuroendocrine differentiation in breast carcinomas has been a matter of discussion since it was first described almost 60 years ago. Indeed, so-called neuroendocrine neoplasms of the breast (Br-NENs) are a less well-defined group of neoplasms than analogous entities in other anatomic sites, such as the lung and the gastroenteropancreatic (GEP) tract. Pure neuroendocrine phenotype is extremely rare, whereas the expression of neuroendocrine markers in usual breast carcinomas, both of special and of non-special type, without evident neuroendocrine morphology, is more common. In this context, the diagnostic criteria and the classification scheme for Br-NENs have been continuously changing over time and real consensus on this topic is still lacking, despite the recent publication of the 5th edition of the WHO classification of breast tumors. In this review, we will recapitulate the evolution of the concept of Br-NEN; revise the available knowledge on their morphological, molecular, and clinical features; and critically discuss the current classification scheme.

Context.— Solid pseudopapillary neoplasm of the pancreas is a low-grade malignant tumor generally associated with a good prognosis. Solid pseudopapillary neoplasms show peculiar morphologic features, but sometimes the differential diagnosis with other pancreatic neoplasms (ie, pancreatic neuroendocrine tumors) can be a challenging task, especially in cytologic or biopsy specimens. In these cases immunohistochemistry is a useful tool, but the diagnostic utility of several proposed immunohistochemical markers is questionable. In recent years, despite several attempts to characterize the pathogenetic, molecular, and prognostic features of solid pseudopapillary neoplasms, they still remain unclear. Objective.— To give the reader a comprehensive update on this entity. Data Sources.— The PubMed database (US National Library of Medicine) was searched using the following string: pseudopapillary tumor [AND/OR] neoplasm [AND/OR] pancreas. All articles written in English were included. In addition, because a heterogeneous terminology has been used in the past to define solid pseudopapillary neoplasms, the reference lists of each paper selected in the PubMed database were also reviewed. Conclusions.— This review gives a comprehensive update on the pathologic, clinical, and molecular features of solid pseudopapillary neoplasms, particularly addressing issues and challenges related to diagnosis. In addition, we have tried to correlate the molecular alterations with the morphologic and clinical features.