ISL1 expression is not restricted to pancreatic well-differentiated neuroendocrine neoplasms, but is also commonly found in well and poorly differentiated neuroendocrine neoplasms of extrapancreatic origin

The human insulin gene enhancer-binding protein islet-1 (ISL1) is a transcription factor involved in the differentiation of the neuroendocrine pancreatic cells. Recent studies identified ISL1 as a marker for pancreatic well-differentiated neuroendocrine neoplasms. However, little is known about ISL1 expression in pancreatic poorly differentiated and in extrapancreatic well and poorly differentiated neuroendocrine neoplasms. We studied the immunohistochemical expression of ISL1 in 124 neuroendocrine neoplasms. Among pancreatic neuroendocrine neoplasms, 12/13 with poor differentiation were negative, whereas 5/7 with good differentiation but a Ki67 >20% were positive. In extrapancreatic neuroendocrine neoplasms, strong positivity was found in Merkel cell carcinomas (25/25), pulmonary small cell neuroendocrine carcinomas (21/23), medullary thyroid carcinomas (9/9), paragangliomas/pheochromocytomas (6/6), adrenal neuroblastomas (8/8) and head and neck neuroendocrine carcinomas (4/5), whereas no or only weak staining was recorded in pulmonary carcinoids (3/15), olfactory neuroblastomas (1/4) and basaloid head and neck squamous cell carcinomas (0/15). ISL1 stained the neuroendocrine carcinoma component of 5/8 composite carcinomas and also normal neuroendocrine cells in the thyroid, adrenal medulla, stomach and colorectum. Poorly differentiated neuroendocrine neoplasms, regardless of their ISL1 expression, were usually TP53 positive. Our results show the almost ubiquitous expression of ISL1 in extrapancreatic poorly differentiated neuroendocrine neoplasms and neuroblastic malignancies and its common loss in pancreatic poorly differentiated neuroendocrine neoplasms. These findings modify the role of ISL1 as a marker for pancreatic neuroendocrine neoplasms and suggest that ISL1 has a broader involvement in differentiation and growth of neuroendocrine neoplasms than has so far been assumed.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

[Computer tomographic differentiation of intrathoracic neoplasms and inflammation in the dog].

The purpose of this study was to elaborate on the advantages and limits of computed tomography (CT) in the differentiation of thoracic lesions, in particular neoplasias. In the course of the investigation CT-scans of the thorax of 31 dogs with lesions in the area of the lungs or mediastinum were evaluated. The lesions were rated by morphology, distribution pattern, attenuation values and contrast-enhancement. Biopsies or the whole body underwent a pathohistological examination. Of the 31 dogs 17 had neoplastic and 14 had inflammatory lesions in the thoracic region. With help of the CT, the exact localisations of the different lesions was possible in most cases. Due to their characteristic morphologies, distribution patterns and attenuation values the differentiation between inflammatory and neoplastic lesions was possible in most cases (n=25/31) on the basis of the CT-scans. Mean non-enhanced CT attenuation values of the neoplastic lesions ranged between 31 and 50 HU, of the inflammatory lesions between -251 and 9 HU. Both neoplastic and inflammatory lesions showed contrast enhancement (between 14 and 38 HU and between 2 and 95 HU respectively). The mediastinal abcesses enhanced mainly on the periphery of the lesion. A differentiation of the various types of neoplastic lesions based on the non-enhanced attenuation values was not successful. Only metastasis could be differentiated because of their distribution pattern.

The modified coronally advanced tunnel combined with an enamel matrix derivative and subepithelial connective tissue graft for the treatment of isolated mandibular Miller Class I and II gingival recessions: a report of 16 cases.

OBJECTIVES To clinically evaluate the healing of mandibular Miller Class I and II isolated gingival recessions treated with the modified coronally advanced tunnel (MCAT) in conjunction with an enamel matrix derivative (EMD) and subepithelial connective tissue graft (SCTG). METHOD AND MATERIALS Sixteen healthy patients (13 women and 3 men) exhibiting one isolated mandibular Miller Class I and II gingival recessions of a depth of ≥ 3 mm, were consecutively treated with the MCAT in conjunction with EMD and SCTG. Treatment outcomes were assessed at baseline and at 12 months postoperatively. The primary outcome variable was complete root coverage (CRC) (eg, 100% root coverage). RESULTS Postoperative pain and discomfort were low and no complications such as postoperative bleeding, allergic reactions, abscesses, or loss of SCTG were observed. At 12 months, statistically significant (P < .0001) root coverage was obtained in all 16 defects. CRC was measured in 12 out of the 16 cases (75%) while in the remaining 4 defects root coverage amounted to 90% (in two cases) and 80% (in two cases), respectively. Mean root coverage was 96.25%. Mean keratinized tissue width increased from 1.98 ± 0.8 mm at baseline to 2.5 ± 0.9 mm (P < .0001) at 12 months, while mean probing depth did not show any statistically significant changes (ie, 1.9 ± 0.3 mm at baseline vs 1.8 ± 0.2 mm at 12 months). CONCLUSION Within their limits, the present results indicate that the described treatment approach may lead to predictable root coverage of isolated mandibular Miller Class I and II gingival recessions.

Regulation of NAMPT in human gingival fibroblasts and biopsies

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.

Prognostic and predictive roles of MGMT protein expression and promoter methylation in sporadic pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS O(6)-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. METHODS We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. RESULTS In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. CONCLUSION Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

[Neuroendocrine neoplasms of the distal jejunum and ileum].

Neuroendocrine neoplasms (NEN) of the distal jejunum and ileum derive from serotonin-producing enterochromaffin (EC) cells. Due to their low proliferation rate and their infiltrative growth, they are often discovered at an advanced disease stage when metastasis has already occurred. The biology of these tumours is different from other NEN of the digestive tract. In order to standardise and improve diagnosis and therapy, the guidelines for the diagnosis and clinical management of jejuno-ileal NEN as well as for the management of patients with liver and other distant metastases from NEN were revised by the European Neuroendocrine Tumour Society (ENETS) in 2012. This review focuses on aspects relevant for surgical pathology.

Implications of Imaging Criteria for the Management and Treatment of Intraductal Papillary Mucinous Neoplasms - Benign versus Malignant Findings

OBJECTIVES Evaluation of computed tomography (CT) and magnetic resonance imaging (MRI) for differentiation of pancreatic intraductal papillary mucinous neoplasm (IPMN) subtypes based on objective imaging criteria. METHODS Fifty-eight patients with 60 histologically confirmed IPMNs were included in this retrospective study. Eighty-three imaging studies (CT,n = 42; MRI,n = 41) were analysed by three independent blinded observers (O1-O3), using established imaging criteria to assess likelihood of malignancy (-5, very likely benign; 5, very likely malignant) and histological subtype (i.e., low-grade (LGD), moderate-grade (MGD), high-grade dysplasia (HGD), early invasive carcinoma (IPMC), solid carcinoma (CA) arising from IPMN). RESULTS Forty-one benign (LGD IPMN,n = 20; MGD IPMN,n = 21) and 19 malignant (HGD IPMN,n = 3; IPMC,n = 6; solid CA,n = 10) IPMNs located in the main duct (n = 6), branch duct (n = 37), or both (n = 17) were evaluated. Overall accuracy of differentiation between benign and malignant IPMNs was 86/92 % (CT/MRI). Exclusion of overtly malignant cases (solid CA) resulted in overall accuracy of 83/90 % (CT/MRI). The presence of mural nodules and ductal lesion size ≥30 mm were significant indicators of malignancy (p = 0.02 and p < 0.001, respectively). CONCLUSIONS Invasive IPMN can be identified with high confidence and sensitivity using CT and MRI. The diagnostic problem that remains is the accurate radiological differentiation of premalignant and non-invasive subtypes. KEY POINTS • CT and MRI can differentiate benign from malignant forms of IPMN. • Identifying (pre)malignant histological IPMN subtypes by CT and MRI is difficult. • Overall, diagnostic performance with MRI was slightly (not significantly) superior to CT.

Diagnosis and workup of 522 consecutive patients with neuroendocrine neoplasms in Switzerland.

BACKGROUND Neuroendocrine neoplasms (NENs) are difficult to diagnose. We used SwissNET data to characterise NEN patients followed in the two academic centres of western Switzerland (WS), and to compare them with patients followed in eastern Switzerland (ES) as well as with international guidelines. METHOD SwissNET is a prospective database covering data from 522 consecutive patients (285 men, 237 women) from WS (n = 99) and ES (n = 423). RESULTS Mean ± SD age at diagnosis was 59.0 ± 15.7 years. Overall, 76/522 experienced a functional syndrome, with a median interval of 1.0 (IQR: 1.0-3.0) year between symptoms onset and diagnosis. A total of 51/522 of these tumours were incidental. The primary tumour site was the small intestine (29%), pancreas (21%), appendix (18%) and lung (11%) in both regions combined. In all, 513 functional imaging studies were obtained (139 in WS, 374 in ES). Of these, 381 were 111In-pentetreotide scintigraphies and 20 were 68Ga-DOTATOC PET. First line therapy was surgery in 87% of patients, medical therapy (biotherapy or chemotherapy) in 9% and irradiation in 3% for both regions together. CONCLUSION Swiss NEN patients appear similar to what has been described in the literature. Imaging by somatostatin receptor scintigraphy (SRS) is widely used in both regions of Switzerland. In good accordance with published guidelines, data on first line therapy demonstrate the crucial role of surgery. The low incidence of biotherapy suggests that long-acting somatostatin analogues are not yet widely used for their anti-proliferative effects. The SwissNET initiative should help improve compliance with ENETS guidelines in the workup and care of NEN patients.

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell-derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33-expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Healing of localized gingival recessions treated with coronally advanced flap alone or combined with either a resorbable collagen matrix or subepithelial connective tissue graft: A preclinical study.

OBJECTIVES To histologically evaluate the effectiveness of a porcine derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for coverage of localized gingival recessions. MATERIALS AND METHODS Chronic single Miller Class I-like recessions were created at the buccal at the canines and at the third and fourth premolars in the upper and lower jaws of six beagle dogs. The defects were randomly treated with (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG, or (3) CAF alone. At 12 weeks, histometric measurements were made, e.g., between a reference point (N) - and the gingival margin (GM) - and the outer contour of the adjacent soft tissue (gingival thickness [GT]). RESULTS The postoperative healing was uneventful in all animals. No complications such as allergic reactions, abscesses or infections were noted throughout the entire study period. All three treatments resulted in coverage of localized gingival recessions. The histological analysis failed to identify any residues of CM or CTG. The histometric measurements revealed comparable outcomes for N-GM and GT values for all three groups (CAF + CM: 1.04 ± 0.69 mm/0.68 ± 0.33 mm; CAF + CTG: 1.15 ± 1.12 mm/0.76 ± 0.37 mm; CAF: 1.43 ± 0.45 mm/0.79 ± 0.24 mm). CONCLUSIONS In the used defect model, the application of CTG or CM in conjunction with CAF did not have an advantage over the use of CAF alone. CLINICAL RELEVANCE The use of CAF alone is a valuable option for the treatment localized Miller Class I recessions.