[Practical application of immunohistochemistry in pancreatic neuroendocrine neoplasms : Tips and pitfalls]. / Praktische Anwendung von Immunhistochemie in pankreatischen neuroendokrinen Neoplasien : Tipps und Pitfalls.

Pancreatic neuroendocrine neoplasms (PanNEN) are rather rare entities. Morphology, combined with immunohistochemistry, allows typing and grading, thereby leading therapeutic decisions. Depending on tumor stage and differential diagnosis, a broad diagnostic panel may be required. The present work summarizes the minimal diagnostic, prognostic, and predictive markers in PanNEN.Markers of choice for defining a neuroendocrine phenotype are synaptophysin, chromogranin A, and INSM1. The proliferation fraction Ki67 is indispensable for grading, while p53 and Rb1 can help in the differentiation from neuroendocrine carcinoma (NEC). Transcription factors, such as cdx2, TTF­1, and Islet­1, can indicate the site of a primary tumor in the setting of a cancer of unknown primary (CUP). DAXX/ATRX immunohistochemistry has mainly prognostic value. Molecular pathology studies currently have little practical value in the diagnosis of PanNEN.An important pitfall in routine diagnostics is the wide spectrum of differential diagnoses mimicking neuroendocrine neoplasms. An expanded immunohistochemical panel is strongly recommended in case of doubt.

RAC1b Collaborates with TAp73α-SMAD4 Signaling to Induce Biglycan Expression and Inhibit Basal and TGF-ß-Driven Cell Motility in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-ß. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-ß pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-ß1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-ß signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-ß1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-ß signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway.

High expression of insulinoma-associated protein 1 (INSM1) distinguishes colorectal mixed and pure neuroendocrine carcinomas from conventional adenocarcinomas with diffuse expression of synaptophysin.

Complementary to synaptophysin and chromogranin A, insulinoma-associated protein 1 (INSM1) has emerged as a sensitive marker for the diagnosis of neuroendocrine neoplasms. Since there are no comparative data regarding INSM1 expression in conventional colorectal adenocarcinomas (CRCs) and colorectal mixed adenoneuroendocrine carcinomas/neuroendocrine carcinomas (MANECs/NECs), we examined INSM1 in a large cohort of conventional CRCs and MANECs/NECs. In conventional CRC, we put a special focus on conventional CRC with diffuse expression of synaptophysin, which carry the risk of being misinterpreted as a MANEC or a NEC. We investigated INSM1 according to the immunoreactive score in our main cohort of 1,033 conventional CRCs and 21 MANECs/NECs in comparison to the expression of synaptophysin and chromogranin A and correlated the results with clinicopathological parameters and patient survival. All MANECs/NECs expressed INSM1, usually showing high or moderate expression (57% high, 34% moderate, and 9% low), which distinguished them from conventional CRCs, which were usually INSM1 negative or low, even if they diffusely expressed synaptophysin. High expression of INSM1 was not observed in conventional CRCs. Chromogranin A was negative/low in most conventional CRCs (99%), but also in most MANECs/NECs (66%). Comparable results were observed in our independent validation cohorts of conventional CRC (n = 274) and MANEC/NEC (n = 19). Similar to synaptophysin, INSM1 expression had no prognostic relevance in conventional CRCs, while true MANEC/NEC showed a highly impaired survival in univariate and multivariate analyses (e.g. disease-specific survival: p < 0.001). MANECs/NECs are a highly aggressive variant of colorectal cancer, which must be reliably identified. High expression of INSM1 distinguishes MANEC/NEC from conventional CRCs with diffuse expression of the standard neuroendocrine marker synaptophysin, which do not share the same dismal prognosis. Therefore, high INSM1 expression is a highly specific/sensitive marker that is supportive for the diagnosis of true colorectal MANEC/NEC.

TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-ß. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-ß/Smad signaling, while preventing non-canonical TGF-ß signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-ß1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73-but not the ß isoform-interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-ß-stimulated pro-invasive ERK activation as an underlying mechanism.

An integrated cellular and molecular model of gastric neuroendocrine cancer evolution highlights therapeutic targets.

Gastric neuroendocrine carcinomas (G-NEC) are aggressive malignancies with poorly understood biology and a lack of disease models. Here, we use genome sequencing to characterize the genomic landscapes of human G-NEC and its histologic variants. We identify global and subtype-specific alterations and expose hitherto unappreciated gains of MYC family members in a large part of cases. Genetic engineering and lineage tracing in mice delineate a model of G-NEC evolution, which defines MYC as a critical driver and positions the cancer cell of origin to the neuroendocrine compartment. MYC-driven tumors have pronounced metastatic competence and display defined signaling addictions, as revealed by large-scale genetic and pharmacologic screening of cell lines and organoid resources. We create global maps of G-NEC dependencies, highlight critical vulnerabilities, and validate therapeutic targets, including candidates for clinical drug repurposing. Our study gives comprehensive insights into G-NEC biology.

Elucidation of the Role of SMAD4 in Epithelial-Mesenchymal Plasticity: Does It Help to Better Understand the Consequences of DPC4 Inactivation in the Malignant Progression of Pancreatic Ductal Adenocarcinoma?

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality worldwide, with a 5-year-survival rate below 10% that is the lowest of all cancer types [...].

Establishment and Molecular Characterization of Two Patient-Derived Pancreatic Ductal Adenocarcinoma Cell Lines as Preclinical Models for Treatment Response.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is exceedingly poor. Although surgical resection is the only curative treatment option, multimodal treatment is of the utmost importance, as only about 20% of tumors are primarily resectable at the time of diagnosis. The choice of chemotherapeutic treatment regimens involving gemcitabine and FOLFIRINOX is currently solely based on the patient's performance status, but, ideally, it should be based on the tumors' individual biology. We established two novel patient-derived primary cell lines from surgical PDAC specimens. LuPanc-1 and LuPanc-2 were derived from a pT3, pN1, G2 and a pT3, pN2, G3 tumor, respectively, and the clinical follow-up was fully annotated. STR-genotyping revealed a unique profile for both cell lines. The population doubling time of LuPanc-2 was substantially longer than that of LuPanc-1 (84 vs. 44 h). Both cell lines exhibited a typical epithelial morphology and expressed moderate levels of CK7 and E-cadherin. LuPanc-1, but not LuPanc-2, co-expressed E-cadherin and vimentin at the single-cell level, suggesting a mixed epithelial-mesenchymal differentiation. LuPanc-1 had a missense mutation (p.R282W) and LuPanc-2 had a frameshift deletion (p.P89X) in TP53. BRCA2 was nonsense-mutated (p.Q780*) and CREBBP was missense-mutated (p.P279R) in LuPanc-1. CDKN2A was missense-mutated (p.H83Y) in LuPanc-2. Notably, only LuPanc-2 harbored a partial or complete deletion of DPC4. LuPanc-1 cells exhibited high basal and transforming growth factor (TGF)-ß1-induced migratory activity in real-time cell migration assays, while LuPanc-2 was refractory. Both LuPanc-1 and LuPanc-2 cells responded to treatment with TGF-ß1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-ß1 target genes, which is consistent with the absence of DPC4 in LuPanc-2 cells. Both cell lines were able to form spheres in a semi-solid medium and in cell viability assays, LuPanc-1 cells were more sensitive than LuPanc-2 cells to treatment with gemcitabine and FOLFIRINOX. In summary, both patient-derived cell lines show distinct molecular phenotypes reflecting their individual tumor biology, with a unique clinical annotation of the respective patients. These preclinical ex vivo models can be further explored for potential new treatment strategies and might help in developing personalized (targeted) therapy regimens.

Design and Transition of an Emergency E-Learning Pathology Course for Medical Students-Evaluation of a Novel Course Concept.

BACKGROUND: Around the world, the emergency brought about by the COVID-19 pandemic forced medical schools to create numerous e-learning supplements to provide instruction during this crisis. The question now is to determine a way in which to capitalize on this momentum of digitization and harness the medical e-learning content created for the future. We have analyzed the transition of a pathology course to an emergency remote education online course and, in the second step, applied a flipped classroom approach including research skills training. METHODS: In the summer semester of 2020, the pathology course at the Technical University of Munich was completely converted to an asynchronous online course. Its content was adapted in winter 2021 and incorporated into a flipped classroom concept in which research skills were taught at the same time. RESULTS: Screencasts and lecture recordings were the most popular asynchronous teaching formats. Students reported developing a higher interest in pathology and research through group work. The amount of content was very challenging for some students. CONCLUSION: Flipped classroom formats are a viable option when using pre-existing content. We recommend checking such content for technical and didactic quality and optimizing it if necessary. Content on research skills can be combined very well with clinical teaching content.

Differential Effects of Somatostatin, Octreotide, and Lanreotide on Neuroendocrine Differentiation and Proliferation in Established and Primary NET Cell Lines: Possible Crosstalk with TGF-ß Signaling.

GEP-NETs are heterogeneous tumors originating from the pancreas (panNET) or the intestinal tract. Only a few patients with NETs are amenable to curative tumor resection, and for most patients, only palliative treatments to successfully control the disease or manage symptoms remain, such as with synthetic somatostatin (SST) analogs (SSAs), such as octreotide (OCT) or lanreotide (LAN). However, even cells expressing low levels of SST receptors (SSTRs) may exhibit significant responses to OCT, which suggests the possibility that SSAs signal through alternative mechanisms, e.g., transforming growth factor (TGF)-ß. This signaling mode has been demonstrated in the established panNET line BON but not yet in other permanent (i.e., QGP) or primary (i.e., NT-3) panNET-derived cells. Here, we performed qPCR, immunoblot analyses, and cell counting assays to assess the effects of SST, OCT, LAN, and TGF-ß1 on neuroendocrine marker expression and cell proliferation in NT-3, QGP, and BON cells. SST and SSAs were found to regulate a set of neuroendocrine genes in all three cell lines, with the effects of SST, mainly LAN, often differing from those of OCT. However, unlike NT-3 cells, BON cells failed to respond to OCT with growth arrest but paradoxically exhibited a growth-stimulatory effect after treatment with LAN. As previously shown for BON, NT-3 cells responded to TGF-ß1 treatment with induction of expression of SST and SSTR2/5. Of note, the ability of NT-3 cells to respond to TGF-ß1 with upregulation of the established TGF-ß target gene SERPINE1 depended on cellular adherence to a collagen-coated matrix. Moreover, when applied to NT-3 cells for an extended period, i.e., 14 days, TGF-ß1 induced growth suppression as shown earlier for BON cells. Finally, next-generation sequencing-based identification of microRNAs (miRNAs) in BON and NT-3 revealed that SST and OCT impact positively or negatively on the regulation of specific miRNAs. Our results suggest that primary panNET cells, such as NT-3, respond similarly as BON cells to SST, SSA, and TGF-ß treatment and thus provide circumstantial evidence that crosstalk of SST and TGF-ß signaling is not confined to BON cells but is a general feature of panNETs.

Suppressive Role of ACVR1/ALK2 in Basal and TGFß1-Induced Cell Migration in Pancreatic Ductal Adenocarcinoma Cells and Identification of a Self-Perpetuating Autoregulatory Loop Involving the Small GTPase RAC1b.

Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor ß1 (TGFß1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFß via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells' migratory activity in the absence or presence of TGFß1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFß1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFß-rich tumor microenvironment.

pT3 colorectal cancer revisited: a multicentric study on the histological depth of invasion in more than 1000 pT3 carcinomas-proposal for a new pT3a/pT3b subclassification.

BACKGROUND: Pathological TNM staging (pTNM) is the strongest prognosticator in colorectal carcinoma (CRC) and the foundation of its post-operative clinical management. Tumours that invade pericolic/perirectal adipose tissue generally fall into the pT3 category without further subdivision. METHODS: The histological depth of invasion into the pericolic/perirectal fat was digitally and conventionally measured in a training cohort of 950 CRCs (Munich). We biostatistically calculated the optimal cut-off to stratify pT3 CRCs into novel pT3a (≤3 mm)/pT3b (>3 mm) subgroups, which were then validated in two independent cohorts (447 CRCs, Bayreuth/542 CRCs, Mainz). RESULTS: Compared to pT3a tumours, pT3b CRCs showed significantly worse disease-specific survival, including in pN0 vs pN+ and colonic vs. rectal cancers (DSS: P < 0.001, respectively, pooled analysis of all cohorts). Furthermore, the pT3a/pT3b subclassification remained an independent predictor of survival in multivariate analyses (e.g. DSS: P < 0.001, hazard ratio: 4.41 for pT3b, pooled analysis of all cohorts). While pT2/pT3a CRCs showed similar survival characteristics, pT3b cancers remained a distinct subgroup with dismal survival. DISCUSSION: The delineation of pT3a/pT3b subcategories of CRC based on the histological depth of adipose tissue invasion adds valuable prognostic information to the current pT3 classification and implementation into current staging practices of CRC should be considered.

Senescence-Associated Molecules and Tumor-Immune-Interactions as Prognostic Biomarkers in Colorectal Cancer.

Background and Aims: The initiation of cellular senescence in response to protumorigenic stimuli counteracts malignant progression in (pre)malignant cells. Besides arresting proliferation, cells entering this terminal differentiation state adopt a characteristic senescence-associated secretory phenotype (SASP) which initiates alterations to their microenvironment and effects immunosurveillance of tumorous lesions. However, some effects mediated by senescent cells contribute to disease progression. Currently, the exploration of senescent cells' impact on the tumor microenvironment and the evaluation of senescence as possible target in colorectal cancer (CRC) therapy demand reliable detection of cellular senescence in vivo. Therefore, specific immunohistochemical biomarkers are required. Our aim is to analyze the clinical implications of senescence detection in colorectal carcinoma and to investigate the interactions of senescent tumor cells and their immune microenvironment in vitro and in vivo. Methods: Senescence was induced in CRC cell lines by low-dose-etoposide treatment and confirmed by Senescence-associated ß-galactosidase (SA-ß-GAL) staining and fluorescence activated cell sorting (FACS) analysis. Co-cultures of senescent cells and immune cells were established. Multiple cell viability assays, electron microscopy and live cell imaging were conducted. Immunohistochemical (IHC) markers of senescence and immune cell subtypes were studied in a cohort of CRC patients by analyzing a tissue micro array (TMA) and performing digital image analysis. Results were compared to disease-specific survival (DSS) and progression-free survival (PFS). Results: Varying expression of senescence markers in tumor cells was associated with in- or decreased survival of CRC patients. Proximity analysis of p21-positive senescent tumor cells and cytotoxic T cells revealed a significantly better prognosis for patients in which these cell types have the possibility to directly interact. In vitro, NK-92 cells (mimicking natural killer T cells) or TALL-104 cells (mimicking both cytotoxic T cells and natural killer T cells) led to dose-dependent specific cytotoxicity in >75 % of the senescent CRC cells but <20 % of the proliferating control CRC cells. This immune cell-mediated senolysis seems to be facilitated via direct cell-cell contact inducing apoptosis and granule exocytosis. Conclusion: Counteracting tumorigenesis, cellular senescence is of significant relevance in CRC. We show the dual role of senescence bearing both beneficial and malignancy-promoting potential in vivo. Absence as well as exceeding expression of senescence markers are associated with bad prognosis in CRC. The antitumorigenic potential of senescence induction is determined by tumor micromilieu and immune cell-mediated elimination of senescent cells.

The Quasimesenchymal Pancreatic Ductal Epithelial Cell Line PANC-1-A Useful Model to Study Clonal Heterogeneity and EMT Subtype Shifting.

Intratumoral heterogeneity (ITH) is an intrinsic feature of malignant tumors that eventually allows a subfraction of resistant cancer cells to clonally evolve and cause therapy failure or relapse. ITH, cellular plasticity and tumor progression are driven by epithelial-mesenchymal transition (EMT) and the reverse process, MET. During these developmental programs, epithelial (E) cells are successively converted to invasive mesenchymal (M) cells, or back to E cells, by passing through a series of intermediate E/M states, a phenomenon termed E-M plasticity (EMP). The induction of MET has clinical potential as it can block the initial EMT stages that favor tumor cell dissemination, while its inhibition can curb metastatic outgrowth at distant sites. In pancreatic ductal adenocarcinoma (PDAC), cellular models with which to study EMP or MET induction are scarce. Here, we have generated single cell-derived clonal cultures of the quasimesenchymal PDAC-derived cell line, PANC-1, and found that these differ strongly with respect to cell morphology and EMT marker expression, allowing for their tentative classification as E, E/M or M. Interestingly, the different EMT phenotypes were found to segregate with differences in tumorigenic potential in vitro, as measured by colony forming and invasive activities, and in circadian clock function. Moreover, the individual clones the phenotypes of which remained stable upon prolonged culture also responded differently to treatment with transforming growth factor (TGF)ß1 in regard to regulation of growth and individual TGFß target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFß1 induced a shift in parental PANC-1 cultures towards a more extreme M and invasive phenotype, while exposing the cells to a combination of the proinflammatory cytokines IFNγ, IL1ß and TNFα (IIT) elicited a shift towards a more E and less invasive phenotype resembling a MET-like process. Finally, we show that the actions of TGFß1 and IIT both converge on regulating the ratio of the small GTPase RAC1 and its splice isoform, RAC1b. Our data provide strong evidence for dynamic EMT-MET transitions and qualify this cell line as a useful model with which to study EMP.

IgG4-related pseudotumours: a series of 12 cases and a review of the literature.

IgG4-related pseudotumours (IgG4-RPT) represent a distinctive manifestation in the broad spectrum of IgG4-related diseases (IgG4-RD). Due to their wide morphology and rarity, IgG4-RPTs represent a diagnostic challenge in the differential between reactive lesions and a fibrous soft tissue tumours. Thus, our aim was to characterise our cases and review the literature, focusing on the macroscopic and microscopic features of the lesions. In this paper, we summarise the possible presentations and histomorphological features of IgG4-RPT based on data collected from the literature and from cases at our institute and provide an overview of the pathogenesis and histological characteristics based on the knowledge accumulated in recent years. We collected surgical cases with a diagnosis of IgG4-RPT over the period 2013-2020 at two centres and analysed their macroscopic, histological, and immunohistochemical profiles. Furthermore, we performed a literature research in the MEDLINE and EBSCO databases regarding case reports and studies with the explicit diagnosis of IgG4-RPT. Our cases consist of nine men and three women, with an average age of 60±14 years, representing about 0.05% of the lesions evaluated at the two departments. The involved sites include the kidney, lung, gallbladder, pterygopalatine fossa, spleen, tongue, mediastinum, and submandibular gland. Grossly, nine lesions showed sharp margins. On histological examination, all the lesions showed an abundant inflammatory infiltrate with lymphocytes and IgG4-positive plasma cells as well as characteristic fibroblastic storiform proliferation. The literature search revealed 266 cases and similar histomorphological features in 23 locations. In 30 of these cases (11%), IgG4-RPTs were multifocal. IgG4-RPT are exceedingly rare lesions, which makes them challenging to diagnose. They can affect different sites, and the histomorphological presentation may differ.

An analysis of 130 neuroendocrine tumors G3 regarding prevalence, origin, metastasis, and diagnostic features.

Limited data exist on high-grade neuroendocrine tumors (NETs G3) which represent a new category among neuroendocrine neoplasms (NEN). We analyzed NETs G3 in a consultation series regarding prevalence, origin, metastasis, and diagnostic problems. Based on the WHO classification of digestive system tumors, 130 NETs G3 (9%) were identified in 1513 NENs. NET G3 samples were more often obtained from metastatic sites (69%) than NET G1/G2 samples (24%). NET G3 metastases presented most frequently in the liver (74%) and originated from the pancreas (38/90, 42%), followed by the lung (9%), ileum (7%), stomach (3%), rectum (1%), and rare sites (2%) such as the prostate and breast. The primaries remained unknown in 15%. NETs G3 had a median Ki67 of 30% that distinguished them from NECs (60%), though with great overlap. The expression of site-specific markers, p53, Rb1, and SST2 was similar in NETs G3 and NETs G1/G2, except for p53 and Rb1 which were abnormally expressed in 8% and 7% of liver metastases from NET G3 but not from NET G1/G2. NETs G3 were frequently referred as NECs (39%) but could be well distinguished from NECs by normal p53 (92% versus 21%) and Rb1 expression (93% versus 41%) expression. In conclusion, NETs G3 are frequently discovered as liver metastases from pancreatic or pulmonary primaries and are often misinterpreted as NEC. p53 and Rb1 are powerful markers in the distinction of NET G3 from NEC. Rarely, carcinomas from non-digestive, non-pulmonary organs with neuroendocrine features may present as NET G3.

Neuroendocrine neoplasms of the pancreas: diagnosis and pitfalls.

Common to neuroendocrine neoplasms of the pancreas is their expression of synaptophysin, chromogranin A, and/or INSM1. They differ, however, in their histological differentiation and molecular profile. Three groups can be distinguished: well-differentiated neuroendocrine neoplasms (neuroendocrine tumors), poorly differentiated neuroendocrine neoplasms (neuroendocrine carcinomas), and mixed neuroendocrine-non-neuroendocrine neoplasms. However, the expression of synaptophysin and, to a lesser extent, also chromogranin A is not restricted to the neuroendocrine neoplasms, but may also be in a subset of non-neuroendocrine epithelial and non-epithelial neoplasms. This review provides the essential criteria for the diagnosis of pancreatic neuroendocrine neoplasms including diagnostic clues for the distinction of high-grade neuroendocrine tumors from neuroendocrine carcinomas and an algorithm avoiding diagnostic pitfalls in the delineation of non-neuroendocrine neoplasms with neuroendocrine features from pancreatic neuroendocrine neoplasms.

Intra-abdominal EWSR1/FUS-CREM-rearranged malignant epithelioid neoplasms: two cases of an emerging aggressive entity with emphasis on misleading immunophenotype.

CREB family (CREB1, ATF1, and CREM) gene fusions are defining markers in diverse mesenchymal neoplasms (clear cell sarcoma, angiomatoid fibrous histiocytoma, and others). However, neoplasms harboring EWSR1-CREM/FUS-CREM fusions are rare and poorly characterized. We describe two cases (55-year-old male with 7.5 cm renal mass and 32-year-old female with 5.5 cm mesenteric mass) illustrating their misleading immunophenotypes. Histologically, both showed eosinophilic and focally clear epithelioid cells arranged into sheets, nests, and trabeculae. Immunohistochemistry showed ALK, EMA, and AE1/AE3 immunoreactivity suggesting ALK-rearranged renal cell carcinoma (Case 1) and coexpression of keratin, EMA, synaptophysin, and chromogranin-A, suggesting neuroendocrine neoplasm (Case 2). Targeted RNA sequencing revealed EWSR1-CREM (Case 1) and FUS-CREM (Case 2) fusions. These cases add to the spectrum of CREM fusion-positive intra-abdominal epithelioid neoplasms. Their unusual immunophenotype and unexpected sites represent major pitfalls, underline a wide differential diagnosis, and emphasize the value of molecular testing in correctly diagnosing them.

The Use of PDX1 DNA Methylation to Distinguish Two Subtypes of Pancreatic Neuroendocrine Neoplasms with Different Prognoses.

Pancreatic neuroendocrine neoplasms (pNENs) account for approximately 5% of all pancreatic tumors; thus, they constitute the second most common tumor type in the pancreas [...].

Loss of SATB2 Occurs More Frequently Than CDX2 Loss in Colorectal Carcinoma and Identifies Particularly Aggressive Cancers in High-Risk Subgroups.

BACKGROUND: Special AT-rich sequence-binding protein 2 (SATB2) has emerged as an alternative immunohistochemical marker to CDX2 for colorectal differentiation. However, the distribution and prognostic relevance of SATB2 expression in colorectal carcinoma (CRC) have to be further elucidated. METHODS: SATB2 expression was analysed in 1039 CRCs and correlated with clinicopathological and morphological factors, CDX2 expression as well as survival parameters within the overall cohort and in clinicopathological subgroups. RESULTS: SATB2 loss was a strong prognosticator in univariate analyses of the overall cohort (p < 0.001 for all survival comparisons) and in numerous subcohorts including high-risk scenarios (UICC stage III/high tumour budding). SATB2 retained its prognostic relevance in multivariate analyses of these high-risk scenarios (e.g., UICC stage III: DSS: p = 0.007, HR: 1.95), but not in the overall cohort (DSS: p = 0.1, HR: 1.25). SATB2 loss was more frequent than CDX2 loss (22.2% vs. 10.2%, p < 0.001) and of higher prognostic relevance with only moderate overlap between SATB2/CDX2 expression groups. CONCLUSIONS: SATB2 loss is able to identify especially aggressive CRCs in high-risk subgroups. While SATB2 is the prognostically superior immunohistochemical parameter compared to CDX2 in univariate analyses, it appears to be the less sensitive marker for colorectal differentiation as it is lost more frequently.

Activation of a Ductal-to-Endocrine Transdifferentiation Transcriptional Program in the Pancreatic Cancer Cell Line PANC-1 Is Controlled by RAC1 and RAC1b through Antagonistic Regulation of Stemness Factors.

Epithelial-mesenchymal transition (EMT) is a driving force for tumor growth, metastatic spread, therapy resistance, and the generation of cancer stem cells (CSCs). However, the regained stem cell character may also be exploited for therapeutic conversion of aggressive tumor cells to benign, highly differentiated cells. The PDAC-derived quasimesenchymal-type cell lines PANC-1 and MIA PaCa-2 have been successfully transdifferentiated to endocrine precursors or insulin-producing cells; however, the underlying mechanism of this increased plasticity remains elusive. Given its crucial role in normal pancreatic endocrine development and tumor progression, both of which involve EMT, we analyzed here the role of the small GTPase RAC1. Ectopic expression in PANC-1 cells of dominant negative or constitutively active mutants of RAC1 activation blocked or enhanced, respectively, the cytokine-induced activation of a ductal-to-endocrine transdifferentiation transcriptional program (deTDtP) as revealed by induction of the NEUROG3, INS, SLC2A2, and MAFA genes. Conversely, ectopic expression of RAC1b, a RAC1 splice isoform and functional antagonist of RAC1-driven EMT, decreased the deTDtP, while genetic knockout of RAC1b dramatically increased it. We further show that inhibition of RAC1 activation attenuated pluripotency marker expression and self-renewal ability, while depletion of RAC1b dramatically enhanced stemness features and clonogenic potential. Finally, rescue experiments involving pharmacological or RNA interference-mediated inhibition of RAC1 or RAC1b, respectively, confirmed that both RAC1 isoforms control the deTDtP in an opposite manner. We conclude that RAC1 and RAC1b antagonistically control growth factor-induced activation of an endocrine transcriptional program and the generation of CSCs in quasimesenchymal PDAC cells. Our results have clinical implications for PDAC patients, who in addition to eradication of tumor cells have a need for replacement of insulin-producing cells.