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Introduction: The mixed adenoneuroendocrine carcinomas (MANEC, WHO 2010) are neoplasms with heterogeneous morphology, whose mixed nature may be difficult to recognize. Aim(s): To increase the awareness of MANECs in colorectal carcinomas with signet-ring cell morphology. Materials and methods: A series of four colorectal carcinomas with signet ring cell component (SRCC) in organoid pattern were studied for neuroendocrine (NE) differentiation. The cases were immunolabeled with antibodies to synaptophysin, Ki-67, p53, glucagon, serotonin, pancreatic-polypeptide (PP), SSTR2, Muc1, Muc2, Muc5a, CDX2, CK20, CK7, CK5/6, E-cadherin and β-catenin. Clinical data are presented. Results: The SRCC made up 40-90% of tumors and was embedded in mucus. Synaptophysin was expressed in >30% (50-65%) of SRCC. The SRCC was G3 in three cases (Ki-67-90, 85 and 60%) and G2 (Ki-67-14%) in one. Muc1, Muc2 and p53 were expressed in all SRCCs. CDX2 and CK20 were positive in 3/4 cases, and CK7 in one. The rest of the stains were negative. A typical NET component was present in two MANECs, one G3 (Ki-67-100%) and one G2 (Ki-67-14%), and an adenoma in other MANEC. All cases with G3-SRCC presented at stage IIIB (1/4) and IVB(2/4), and the MANEC with G2-SRCC at stage I. All were nonfunctional and most received chemotherapy, with partial response up to now. Conclusion: Colorectal adenocarcinomas with signet-ring cell morphology may turn out to be MANECs according to the WHO classification 2010, if carefully screened for neuroendocrine differentiation. [ABSTRACT FROM AUTHOR]

Neuroendocrine neoplasms (NENs) encompass a heterogeneous spectrum of tumors originating from the diffuse neuroendocrine cell system. Approximately 30% of NEN exhibit functional activity with clinical syndromes through hormone-mediated effects. Synchronous and metachronous functioning syndromes, resulting from the simultaneous release of distinct hormones, are exceptionally rare. Of note, hormonal excess syndromes can have a greater effect on patients' morbidity and mortality than the tumor mass itself. We present the case of a 49-year-old male patient affected by an oligo-metastatic ileal NEN, concurrently demonstrating vasointestinal peptide (VIP) and serotonin excretion, complicated by pulmonary tuberculosis. After the first cycle of Lutetium-177-DOTATATE peptide-radio-receptor therapy, the patient developed a severe watery diarrhea, hypokalemia, and achlorhydria (WDHA) syndrome, despite receiving high-dose somatostatin analogues, everolimus, and telotristat ethyl, without any surgical options. The WDHA syndrome necessitated intensive-care-unit (ICU) admission with continual intravenous administration of electrolytes and fluids. With limited alternatives, an off-label intervention using the enkephalinase inhibitor racecadotril was initiated. After 5 days of treatment, the WDHA syndrome exhibited sufficient control, facilitating the patient's discharge from the ICU. This case report underscores racecadotril as an individualized, off-label treatment strategy for patients with severe VIPoma and serotonin-driven WDHA syndrome, where conventional therapeutic avenues have been exhausted., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Tenosynovial giant cell tumor (TGCT) is a rare type of tumor that originates from the synovium of joints and tendon sheaths. It is characterized by recurring genetic abnormalities, often involving the CSF1 gene. Common symptoms include pain and swelling, which are not specific to TGCT, so MRI and a pathological biopsy are needed for an accurate diagnosis. We report the case of a 45-year-old man who experienced painful swelling in his right hip for six months. Initially, this was diagnosed as Erdheim-Chester disease. However, whole exome sequencing (WES) and RNA-Sequencing revealed a CSF1::GAPDHP64 fusion, leading to a revised diagnosis of TGCT. The patient was treated with pegylated interferon and imatinib, which resulted in stable disease after three months. Single-cell transcriptome analysis identified seven distinct cell clusters, revealing that neoplastic cells expressing CSF1 attract macrophages. Analysis of ligand-receptor interactions showed significant communication between neoplastic cells and macrophages mediated by CSF1 and CSF1R. Our findings emphasize the importance of comprehensive molecular analysis in diagnosing and treating rare malignancies like TGCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Fähnrich, Gasimova, Maluje, Ott, Sievert, Fliedner, Reimer, Künstner, Gebauer, Kebenko, von Bubnoff, Kirfel, Sailer, Röcken, Konukiewitz, Klapper, Frydrychowicz, Mogadas, Huebner, Busch and Khandanpour.)

Neuroendocrine neoplasms are classified according to the WHO classification based on morphological criteria into neuroendocrine tumors, neuroendocrine carcinomas, and mixed neuroendocrine-non-neuroendocrine neoplasms. Neuroendocrine tumors are well differentiated neoplasms and show characteristic site-specific histological and molecular features, which is important for their clinical management. In cases dealing with metastasis, pathology often can help to identify the primary tumors using a small immunohistochemical marker panel. Neuroendocrine carcinomas are poorly differentiated neoplasms. They are subdivided into neuroendocrine carcinomas of small cell and large cell type. The molecular profile of neuroendocrine carcinomas and mixed neuroendocrine-non-neuroendocrine neoplasms shows a close relationship to conventional adenocarcinomas with site-specific features. Molecular analysis of neuroendocrine carcinomas and neuroendocrine-non-neuroendocrine neoplasms are not yet fully integrated in daily diagnostics and are mainly performed in the context of precision oncology., Competing Interests: Prof. Dominik Schulte hat Honorare für Vortragstätigkeiten von Ipsen und Novartis erhalten. Prof. Björn Konukiewitz hat Honorare für Vortragstätigkeiten non Novartis erhalten., (Thieme. All rights reserved.)

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, TTF‑1, and Islet‑1, 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., (© 2024. The Author(s).)

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)-β. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-β 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-β1 on BGN . TAp73-mediated regulation of BGN , and inhibition of TGF-β 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-β1-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-β 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.

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., (© 2023 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

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)-β. 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-β/Smad signaling, while preventing non-canonical TGF-β 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-β1-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 β 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-β-stimulated pro-invasive ERK activation as an underlying mechanism.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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)-β1-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-β1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-β1 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.

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.

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)-β. 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-β1 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-β1 treatment with induction of expression of SST and SSTR2 / 5 . Of note, the ability of NT-3 cells to respond to TGF-β1 with upregulation of the established TGF-β 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-β1 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-β treatment and thus provide circumstantial evidence that crosstalk of SST and TGF-β signaling is not confined to BON cells but is a general feature of panNETs.

Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ 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 TGFβ1 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 TGFβ1-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 TGFβ-rich tumor microenvironment.

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., (© 2022. The Author(s).)

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., (Copyright © 2022 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

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)β1 in regard to regulation of growth and individual TGFβ target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFβ1 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, IL1β 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 TGFβ1 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.

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 β-galactosidase (SA-β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kellers, Fernandez, Konukiewitz, Schindeldecker, Tagscherer, Heintz, Jesinghaus, Roth and Foersch.)

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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

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.

Background: Immunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin-eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters., Methods: We evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups., Results: CDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P < 0.001) and showed worse survival characteristics in the overall cohort/UICC Stage II/III (e.g. DFS: P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor., Conclusion: CDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors., (© 2021. The Author(s).)

Mimickers of neuroendocrine neoplasms (NEN) include a number of important pitfall tumors. Here, we describe our experience with mesenchymal mimics of NENs to illustrate their spectrum and draw the attention particularly to a group of mesenchymal/non-epithelial neoplasms (MN) that combine epithelioid histology with neuroendocrine (NE-) features and peculiar genetic abnormalities. In a consultation series of 4498 cases collected between 2009 and 2021, 2099 neoplasms expressing synaptophysin and/or chromograninA were reviewed and analyzed. A total of 364 (18%) were diagnosed as non-NENs, while the remaining tumors were NEN. The group of mesenchymal/non-epithelial neoplasms with NE-features (MN-NE) included 31/364 (8%) cases. These mostly malignant neoplasms showed an epithelioid morphology. While all but one tumor expressed synaptophysin, mostly patchy, only 10/29 (34%) co-expressed chromograninA. A total of 13/31 (42%) of the MN-NE showed EWSR1-related gene fusions (6 Ewing sarcomas, 5 clear cell sarcomas, and 1 desmoplastic small round cell tumor, 1 neoplasm with FUS-CREM gene fusion) and 7 (23%) were SWI/SNF (SMARCB1 or SMARCA4)-deficient neoplasms. The remaining MN-NE included synovial sarcoma, sclerosing epithelioid mesenchymal neoplasm, melanoma, alveolar soft part sarcoma, solitary fibrous tumor, and chordoma. A total of 27/31 MN-NE were from the last 8 years, and 6 of them were located in the pancreas. Eleven MN-NE were initially diagnosed as neuroendocrine carcinomas (NECs). MN-NE with epithelioid features play an increasing role as mimickers of NECs. They mostly belong to tumors with gene fusions involving the EWSR1 gene, or with SWI/SNF complex deficiency. Synaptophysin expression is mostly patchy and chromograninA expression is infrequent in MN-NE of this series and data extracted from literature., (© 2021. The Author(s).)

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.

Background: Colorectal mixed adenoneuroendocrine carcinomas (MANECs) are clinically highly aggressive neoplasms. MANECs are composed of variable adenocarcinoma components combined with morphologically distinct neuroendocrine carcinoma components, which are confirmed by synaptophysin immunohistochemistry, the gold standard marker of a neuroendocrine differentiation. However, the biological behavior of adenocarcinomas that express synaptophysin but do not show a typical neuroendocrine morphology remains unclear., Methods: We investigated synaptophysin expression in 1002 conventional colorectal adenocarcinomas and correlated the results with clinicopathological characteristics and patient survival and compared the survival characteristics of synaptophysin expression groups to MANECs., Results: Synaptophysin expression in conventional colorectal adenocarcinomas was associated with a shortened disease-free survival ( p = 0.037), but not with overall survival or disease-specific survival (DSS) in univariate analyses and without any survival impact in multivariate analyses. Patients with "true" MANECs, on the other hand, showed a significantly shorter survival than all conventional adenocarcinomas with or without synaptophysin expression in uni- and multivariate analyses (e.g., multivariate DSS: p < 0.001, HR: 5.20)., Conclusions: Our study demonstrates that synaptophysin expression in conventional colorectal adenocarcinomas, in contrast to MANECs, is not associated with a significantly poorer clinical outcome when compared to adenocarcinomas without synaptophysin expression. Furthermore, our data suggest that conventional adenocarcinomas with a diffuse synaptophysin expression should not be classified as MANECs, also strongly arguing that synaptophysin testing should be reserved for carcinomas with an H&E morphology suggestive of a neuroendocrine differentiation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and therapy-resistant cancer types which is largely due to tumor heterogeneity, cancer cell de-differentiation, and early metastatic spread. The major molecular subtypes of PDAC are designated classical/epithelial (E) and quasi-mesenchymal (QM) subtypes, with the latter having the worst prognosis. Epithelial-mesenchymal transition (EMT) and the reverse process, mesenchymal-epithelial transition (MET), are involved in regulating invasion/metastasis and stem cell generation in cancer cells but also early pancreatic endocrine differentiation or de-differentiation of adult pancreatic islet cells in vitro, suggesting that pancreatic ductal exocrine and endocrine cells share common EMT programs. Using a panel of PDAC-derived cell lines classified by epithelial/mesenchymal expression as either E or QM, we compared their trans-differentiation (TD) potential to endocrine progenitor or β cell-like cells since studies with human pancreatic cancer cells for possible future TD therapy in PDAC patients are not available so far. We observed that QM cell lines responded strongly to TD culture using as inducers 5'-aza-2'-deoxycytidine or growth factors/cytokines, while their E counterparts were refractory or showed only a weak response. Moreover, the gain of plasticity was associated with a decrease in proliferative and migratory activities and was directly related to epigenetic changes acquired during selection of a metastatic phenotype as revealed by TD experiments using the paired isogenic COLO 357-L3.6pl model. Our data indicate that a QM phenotype in PDAC coincides with increased plasticity and heightened trans-differentiation potential to activate a pancreatic β cell-specific transcriptional program. We strongly assume that this specific biological feature has potential to be exploited clinically in TD-based therapy to convert metastatic PDAC cells into less malignant or even benign cells.

Ductal adenocarcinoma is the most common tumor of the pancreas. Although relatively rare, it poses one of the greatest oncological challenges because of its poor prognosis, which has so far only slightly improved. Progress has been made in the more precise classification and standardization of the morphological assessment. In the current WHO classification, prognostically relevant subtypes are clearly delimited among themselves and from ductal adenocarcinoma not otherwise specified (NOS). In the recent TNM classification, a size-based T‑category was introduced. Diagnostically, the histological assessment of the resection specimen is relatively easy; on the other hand, assessment of the fine-needle biopsy from the primary tumor or a liver metastasis is still difficult. The molecular stratification of ductal adenocarcinoma and the other pancreatic neoplasms has made great progress. This not only defined the genetics of tumor entities, but also identified the prognosis and biology of tumor groups on the basis of RNA expression patterns. The range of treatment could be expanded by targeted molecular therapies (especially for patients with BRCA1/2 germline mutations, NTRK- or NRG1-fusions, or oncogenic BRAF and PIK3CA mutations as well as tumors with microsatellite instability (MSI)), even if targeted therapies are currently only available for a minority of patients (<10%)., (© 2021. Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The 2019 World Health Organization (WHO) classification of colorectal carcinoma (CRC) profoundly reclassified CRC subtypes and introduces tumor budding as a second major grading criterion, while condensing conventional grade into a 2-tiered system. So far it remains largely unexplored how these parameters interact with each other and whether they truly have an independent impact on patient prognosis. We reclassified a large single-center cohort of 1004 CRCs spanning 2 decades for adjusted WHO grade (low vs. high), tumor budding (Bd1/Bd2/Bd3), and CRC subtype (adenocarcinoma not otherwise specified, micropapillary, mucinous, serrated, medullary, adenoma-like, signet-ring cell, mixed adenoneuroendocrine carcinoma/neuroendocrine carcinoma, undifferentiated) according to the criteria of the 2019 WHO classification. We investigated the interaction of these parameters, their connection to stage/microsatellite status, and their significance for patient survival in the different subgroups. Specific subtypes other than adenocarcinoma not otherwise specified represented one third of all CRCs and were unevenly distributed throughout stage and microsatellite subgroups. Subtypes, WHO grade and tumor budding profoundly impacted all survival parameters (P<0.001 for all analyses), with CRC subtypes and tumor budding-but not WHO grade-being stage-independent prognosticators for all survival comparisons. WHO grade had very limited prognostic value in CRC subtypes, while tumor budding retained its strong prognostic impact in most scenarios. Accurate delineation of CRC subtypes introduced in the 2019 WHO classification provides strong stage-independent prognostic information, arguing that they should be considered in pathology reports and in clinical trials. Of the morphology-based grading schemes included in the 2019 WHO, tumor budding outperforms WHO grade., Competing Interests: Conflicts of Interest and Source of Funding: Supported by grants of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation): Project-ID 360372040—SFB 1335 to W.W. and K.S. Project-ID 39535707—SFB 1371. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Tumor regression grading (TRG) based on histopathology is the main tool to assess therapy effects after neoadjuvant therapy (NAT) of pancreatic ductal adenocarcinoma (PDAC). However, reliable markers to distinguish therapy effects from pre-existing tumor features are lacking. The aim of this study was the characterization of PDAC after NAT, focusing on the stroma., Material and Methods: Tissue samples from patients resected for PDAC after NAT (n=27) were analyzed. TRG was assessed using the Royal North Shore (RNS) system. Stromal composition was evaluated by Movat's stain. Immunohistochemistry (IH) for Ki-67 and five previously established stroma markers (alpha-Crystallin B, alpha-Smooth muscle actin (alpha-SMA), Neurotrophin-3 (NT-3), SPARC and Tenascin C) was also performed. Results were compared with therapy-naïve PDACs (n=10)., Results: Most cases showed a moderate response (RNS 2; 74%), while 15% displayed a poor response (RNS 3), and 11% a good response (RNS 1). No complete response was observed. Poor regression was associated with mucin-rich stroma, while good regression was associated with collagen-rich stroma. Cases with poorer therapy response had significantly higher proliferation. Higher peritumoral staining intensity for alpha-SMA and Tenascin C also showed a trend towards an association with poor regression., Conclusions: Similar to the stroma in therapy-naïve PDAC, the stroma of PDAC after NAT is heterogeneous. Distinguishing between desmoplastic stroma and therapy-induced fibrosis by single markers is not possible. Movat's pentachrome stain, IH for Ki-67, and to some extent for Tenascin C and alpha-SMA, can help detect poor histopathological response to NAT.

The molecular carcinogenesis of intraductal tubulopapillary neoplasms (ITPN), recently described as rare neoplasms in the pancreato-biliary tract with a favorable prognosis despite a high incidence of associated pancreato-biliary adenocarcinoma, is still poorly understood. To identify driver genes, chromosomal gains and losses, mutational signatures, key signaling pathways, and potential therapeutic targets, the molecular profile of 11 biliary and 6 pancreatic ITPNs, associated with invasive adenocarcinoma in 14/17 cases, are studied by whole exome sequencing (WES). The WES of 17 ITPNs reveals common copy number variants (CNVs) broadly distributed across the genome, with recurrent chromosomal deletions primarily in 1p36 and 9p21 affecting the tumor suppressors CHD5 and CDKN2A , respectively, and gains in 1q affecting the prominent oncogene AKT3 . The identified somatic nucleotide variants (SNVs) involve few core signaling pathways despite high genetic heterogeneity with diverse mutational spectra: Chromatin remodeling, the cell cycle, and DNA damage/repair. An OncoKB search identifies putative actionable genomic targets in 35% of the cases (6/17), including recurrent missense mutations of the FGFR2 gene in biliary ITPNs (2/11, 18%). Our results show that somatic SNV in classical cancer genes, typically associated with pancreato-biliary carcinogenesis, were absent ( KRAS , IDH1/2 , GNAS , and others) to rare ( TP53 and SMAD4 , 6%, respectively) in ITPNs. Mutational signature pattern analysis reveals a predominance of an age-related pattern. Our findings highlight that biliary ITPN and classical cholangiocarcinoma display commonalities, in particular mutations in genes of the chromatin remodeling pathway, and appear, therefore, more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma.

Background: Ecthyma gangrenosum (EG) is a cutaneous infectious disease characterized by eschar-like skin ulcers typically caused by Pseudomonas aeruginosa. Here, we report a case of relapsing EG in a patient who had returned from a trip to Colombia, thus establishing EG as an important differential diagnosis of tropical diseases, and demonstrating that even long-term antibiotic treatment can result in only partial remission of EG., Case Presentation: A 77-year-old man with underlying chronic lymphocytic leukemia (CLL) on ibrutinib treatment was admitted because of a superinfected mosquito bite on the left ear and multiple partially necrotic skin lesions disseminated all over the entire body five days after returning from a trip to Colombia. The initial clinical suspicion of a tropical disease (leishmaniosis, systemic mycosis, or others) could not be confirmed. During the diagnostic workup, microbiological cultures of the skin biopsies and bronchoalveolar lavage revealed Pseudomonas aeruginosa, leading to a diagnosis of EG. Initial antibiotic treatment resulted in partial remission. However, the patient had to be re-admitted due to a relapse 3-4 weeks after the first episode. Finally, the patient was successfully treated with a combined approach consisting of antibiotics, recurrent surgical incisions, and administration of immunoglobulins., Conclusions: In conclusion, EG should be considered as a differential diagnosis in immunosuppressed patients presenting with eschar-like skin ulcers. A combined treatment approach seems to be the best choice to achieve clinical cure and avoid relapse.

Although canine pancreatic neuroendocrine neoplasms (PanNENs) have been proposed as a model for the counterpart human neoplasms, the type or grade of human PanNEN that they resemble is unclear. PanNENs in animals are classified as adenoma or carcinoma, whereas in humans they are classified as pancreatic neuroendocrine tumour (PanNET) if well-differentiated, or as pancreatic neuroendocrine carcinoma (PanNEC) if poorly differentiated. We evaluated 16 canine primary PanNENs and two metastases histologically and immunohistochemically, and graded them using the animal and human grading systems. All neoplasms had local or vascular invasion and were classified as pancreatic islet cell carcinomas according to the current WHO classification. The Ki-67 index was low in all cases (0.01-1.50%). All had cytoplasmic expression of synaptophysin and insulin but were immunonegative for glucagon, confirming a functional diagnosis of canine insulinoma. Membranous expression of SSTR2A and nuclear expression of ATRX, but no p53 expression, was found in all neoplasms. One primary tumour was diagnosed as a mixed neuroendocrine-non-neuroendocrine neoplasm, which is the first report of this neoplasm in dogs. The other 15 primary tumours and both metastatic tumours were graded as PanNET G1, according to the human WHO classification. We conclude that canine PanNENs share well-differentiated histomorphology, SSTR2A expression and absence of nuclear p53 immunolabelling with human PanNETs G1. However, they differ in ATRX gene expression and functionality, and seem to have a worse prognosis than human PanNETs G1, although their generally low Ki-67 index precludes more precise assessment of prognosis. Membranous SSTR2A expression renders canine PanNENs potentially amenable to treatment with somatostatin analogues or SSTR targeted in-vivo imaging methods., Competing Interests: Conflict of interest statement The authors declare no conflicts of interest in relation to the research, authorship or publication of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A small fraction of pancreatic neuroendocrine tumors (PanNETs) shows a solid, paraganglioma-like (PG-like) histology. We wanted to know whether these PanNETs have a special hormone expression and are related to paragangliomas (PGs)/pheochromocytomas (PCs). We screened a series of 48 surgically resected PanNETs for their histological growth patterns and their association with expression of islet hormones. The PanNETs were divided into PG-like and non-PG-like tumors and immunohistochemically monitored for the expression of islet hormones, cytokeratins, and S100. The results were correlated to histological pattern, lymph node status, and data in 28 PGs/PCs, including 2 PGs attached to the pancreas. All PanNETs, in contrast to PGs/PCs, were cytokeratin positive. A PG-like growth pattern was identified in 9 of 48 PanNETs and correlated with somatostatin expression. Only half of the non-PG-like PanNETs also contained somatostatin-positive cells. Eight of 28 PGs/PCs expressed somatostatin, mostly in individual cells. PG-like PanNETs and non-PG-like PanNETs infiltrated the adjacent pancreatic tissue, whereas 2 to the pancreas-associated PGs were well demarcated. Lymph node metastases were detected in 58%, 39%, 57%, and 53% of the somatostatin-producing, somatostatin-negative, PG-like, and non-PG-like PanNETs, respectively. PG-like PanNETs, in contrast to PG/PCs, are characterized by the expression of cytokeratin and somatostatin, the development of lymph node metastasis, and the infiltration into pancreatic parenchyma. Non-PG-like PanNETs may also express somatostatin and show lymph node metastases to the same extent. A literature review of cases reported as PG of the pancreas reveals that only a small fraction of these tumors probably represents true pancreatic PGs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Pre-operative treatment planning in head and neck squamous cell carcinoma (HNSCC) is mainly dictated by clinical staging, which has major shortcomings. Histologic grading is irrelevant due to its lack of prognostic impact. Recently, a novel grading termed Cellular Dissociation Grade (CDG) based on Tumour Budding and Cell Nest Size was shown to be highly prognostic for resected HNSCC. We aimed to probe the predictive and prognostic impact of CDG in the pre-operative biopsies of HNSCC., Methods: We evaluated CDG in n = 160 pre-therapeutic biopsies from patients who received standardised treatment following German guidelines, and correlated the results with pre- and post-therapeutic staging data and clinical outcome., Results: Pre-operative CDG was highly predictive of post-operative tumour stage, including the prediction of occult lymph node metastasis. Uni- and multivariate analysis revealed CDG to be an independent prognosticator of overall, disease-specific and disease-free survival (p < 0.001). Hazard ratio for disease-specific survival was 6.1 (11.1) for nG2 (nG3) compared with nG1 tumours., Conclusions: CDG is a strong outcome predictor in the pre-treatment scenario of HNSCC and identifies patients with nodal-negative disease. CDG is a purely histology-based prognosticator in the pre-therapeutic setting that supplements clinical staging and may aide therapeutic stratification of HNSCC patients.

Purpose: Neoadjuvant therapy (neoTx) has dramatically improved the prognosis of patients with locally advanced and borderline resectable pancreatic ductal adenocarcinoma, yet its mechanisms of action on tumor cells and the tumor microenvironment are still unknown. Here, we aimed to characterize the multiple facets of neoTx-induced alterations in the pancreatic cancer microenvironment., Experimental Design: We performed the currently most comprehensive histopathologic analysis of desmoplasia, angiogenesis, neural invasion, and immune cell infiltration at the tumor-host interface of pancreatic cancer after neoTx ( n = 37) versus after primary resection ( n = 37) through quantitative IHC and double immunofluorescence using automated and software-based quantification algorithms., Results: We demonstrate that, independently of the applied pretreatment, neoadjuvant regimes are able to reverse the immunosuppressive behavior of malignant cells on pancreatic cancer microenvironment. Here, neoTx-driven selective depletion of regulatory T cells and myeloid-derived suppressor cells was associated with enrichment of antitumor immune cells in the peritumoral niche, decreased stromal activation, and less neural invasion. Importantly, the degree of this antitumor immune remodeling correlates to the degree of histopathologic response to neoTx. Survival analysis revealed that the tumor proliferation rate together with the activation of the stroma and the intratumoral infiltration with CD4 + T cells and natural killer cells constitute as independent prognostic factors for neoadjuvantly treated pancreatic cancer., Conclusions: NeoTx is not only cytotoxic but has pleiotropic, beneficial effects on all cellular and noncellular components of pancreatic cancer. Combinational approaches including immunotherapy may unleash long-term and more effective antitumor responses and improve prognosis of pancreatic cancer., (©2019 American Association for Cancer Research.)