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Studies comparing EUS-guided fine-needle aspiration (EUS-FNA) with EUS-guided fine-needle biopsy (EUS-FNB) for the evaluation of pancreatic neuroendocrine tumors (pNETs) are lacking. We aimed at comparing EUS-FNA with EUS-FNB in terms of Ki-67 proliferative index (PI) estimation capability, cellularity of the samples, and reliability of Ki-67 PI/tumor grading compared with surgical specimens.Patients diagnosed with pNETs on EUS and/or surgical specimens were retrospectively identified. Specimens were re-evaluated to assess Ki-67 PI feasibility, sample cellularity by manual counting, and determination of Ki-67 PI value. Outcomes in the EUS-FNA and EUS-FNB groups were compared. Kendall rank test was used for Ki-67 PI correlation between EUS and surgical specimens. Subgroup analysis including small (≤20 mm), non-functioning pNETs was performed.Three-hundred samples from 292 lesions were evaluated: 69 EUS-FNA cytology and 231 EUS-FNB histology. Ki-67 PI feasibility was similar for EUS-FNA and EUS-FNB (91.3% vs. 95.7%, p = 0.15), while EUS-FNB performed significantly better in the subgroup of 179 small pNETs (88.2% vs. 96.1%, p = 0.04). Rate of poor cellulated (500 cells) specimens was equal between EUS-FNA and EUS-FNB. A significant correlation for Ki-67 PI values between EUS and 92 correspondent surgical specimens was found in both groups, but it was stronger with EUS-FNB (tau = 0.626, p 0.0001 vs. tau = 0.452, p = 0.031). Correct grading estimation was comparable between the two groups (p = 0.482).Our study showed stronger correlation for Ki-67 values between EUS-FNB and surgical specimens, and that EUS-FNB outperformed EUS-FNA in the evaluation of small pNETs. EUS-FNB should become standard of care for grading assessment of suspected pNETs.

Introduction: The combined use of 68gallium (68Ga)-DOTA-peptides and 18fluorine-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) scans in the workup of pancreatic neuroendocrine tumors (PanNETs) is controversial. This study aimed at assessing both tracers’ capability to identify tumors and to assess its association with pathological predictors of recurrence. Methods: Prospectively collected, preoperative, dual-tracer PET/CT scan data of G1-G2, nonmetastatic, PanNETs that underwent surgery between January 2013 and October 2019 were retrospectively analyzed. Results: The final cohort consisted of 124 cases. There was an approximately equal distribution of males and females (50.8%/49.2%) and G1 and G2 tumors (49.2%/50.8%). The disease was detected in 122 (98.4%) and 64 (51.6%) cases by 68Ga-DOTATOC and by 18F-FDG PET/CT scans, respectively, with a combined sensitivity of 99.2%. 18F-FDG-positive examinations found G2 tumors more often than G1 (59.4 vs. 40.6%; p = 0.036), and 18F-FDG-positive PanNETs were larger than negative ones (median tumor size 32 mm, interquartile range [IQR] 21 vs. 26 mm, IQR 20; p = 0.019). The median Ki67 for 18F-FDG-positive and -negative examinations was 3 (IQR 4) and 2 (IQR 4), respectively (p = 0.029). At least 1 pathological predictor of recurrence was present in 74.6% of 18F-FDG-positive cases (vs. 56.7%; p = 0.039), whereas this was not found when dichotomizing the PanNETs by their dimensions (≤/>20 mm). None of the 2 tracers predicted nodal metastasis. The receiver operating characteristic curve analysis showed that 18F-FDG uptake higher than 4.2 had a sensitivity of 49.2% and specificity of 73.3% for differentiating G1 from G2 (AUC = 0.624, p = 0.009). Conclusion: The complementary adoption of 68Ga-DOTATOC and 18F-FDG tracers may be valuable in the diagnostic workup of PanNETs despite not being a game-changer for the management of PanNETs ≤20 mm.

Hepatoid tumors (HTs) represent a rare group of neoplasms that are histologically similar to hepatocellular carcinoma but arise outside the liver. The current World Health Organization classification recognizes the hepatoid morphology of pancreatic tumors only as a possible variant of pancreatic ductal adenocarcinoma (PDAC). Here, we describe two cases of ""pure"" HT of the pancreas showing common features and characterized by indolent biological behavior. These tumors were roundish nodules with pushing borders, hyaline globules, and pure hepatoid histology; they were diffusely positive for beta-catenin and LEF1 on immunohistochemistry. At next-generation sequencing, both neoplasms harbored only one pathogenic somatic mutation that affected the CTNNB1 gene at exon 3 and showed a loss of heterozygosity on chromosomes 18 and 21. By integrating macroscopic and microscopic features, along with their molecular profiles, we advocate that such tumors represent a distinct entity from PDAC and should be considered a new variant of solid pseudopapillary neoplasms. The recognition of this new neoplastic category may have immediate implications not only for tumor taxonomy but also for clinical practice., Universita degli Studi di Verona; CRUI-CARE; Associazione Italiana Ricerca sul Cancro; Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi”; Fondazione Italiana Malattie Pancreas (FIMP)

Objective Acinar cell carcinoma (ACC) of the pancreas are known to be rare and difficult to be recognize because they mimic other unrelated tumors (neuroendocrine, solid pseudopapillary) with different clinical behavior. Especially in the setting of inoperable patients, fine needle aspiration cytology (FNAC), core needle biopsy (FNAB) and immunocyto/histochemistry (ICC/IHC) play a crucial role in the differential diagnosis. The biological material available for ICC tests obtained by minimal invasive procedures is usually limited. Aim of the current study was to evaluate diagnostic panel based on a limited number of ICC markers for typing preoperatively ACC of the pancreas. Methods Of 1820 needle sampling procedures performed and related to pancreatic lesions, 21 cases were extracted with a confirmed diagnosis of ACC on histology. Of them,12 were pure ACC and 9 mixed acinar-neuroendocrine carcinoma (MANEC). Smears of ACC, MANEC and a control group composed of 34neuroendocrine, 7solid pseudopapillary, 50ductal and 4 adenosquamous carcinoma were assessed with an ICC panel made up of BCL10, trypsin, synaptophysin, chromograninA, β-catenin. Results On cytology, BCL10 sensitivity and specificity for ACC was 100%. Trypsin correctly recognized 90% of the cases. Synaptophysin was helpful to correctly identify all the cases with a mixed neuroendocrine component. No significant cross-reaction was observed between BCL10 and trypsin in any of the control group case. Conclusions BCL10 is a determinant marker for the diagnosis of acinar cell carcinoma and mixed acinar neuroendocrine cell carcinoma of the pancreas in a pre-operative citologic/histologic setting.

Thrombotic microangiopathies (TMAs) include a heterogeneous group of diseases characterized by abnormalities in the vessel walls of arterioles and capillaries resulting in microvascular thrombosis that typically presents with a microangiopathic hemolytic anemia (MAHA) and severe thrombocytopenia. We describe here the case of an 82-year-old woman, who came to our attention for a clinical condition consistent with thrombotic microangiopathy. Even if initially highly suggestive for a thrombotic thrombocytopenic purpura (TTP), the elevated ADAMTS13 activity together with the alteration of the main coagulation parameters (D-dimer elevation, fibrinogen consumption, slightly prolonged prothrombin time), induced us to consider several other diseases in the differential diagnostic process. The case evolved toward a suspected overlapped secondary hemophagocytic syndrome, though the hyperferritinemia was finally interpreted within the frame of a cytokine storm. After a complex diagnostic workup, the clinical and biochemical parameters guided us toward the diagnosis of a cancer-related microangiopathic hemolytic anemia (CR-MAHA) secondary to a relapsing breast cancer with multiple metastatic localizations. Prednisone 1 mg/kg body weight was started, and several units of fresh frozen plasma were infused, obtaining a good control of the hemolysis. No specific oncological therapies were, however, possible, due to the older age and the critically compromised general condition of the patient; therefore, after clinical stabilization, the patient was discharged for treatment in a palliative care Hospital.

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream ofGremlin 1(GREM1), which was associated with increasedGREM1expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications., The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.

Purpose: We assessed the frequency of triple-positive serology (viral capsid antigen [VCA]–immunoglobulin G [IgG], VCA–immunoglobulin M, Epstein-Barr nuclear antigen–IgG) for Epstein-Barr virus (EBV) in a small number of patients with angioimmunoblastic T-cell lymphoma (AITL) at disease onset. Methods: Nine patients with newly diagnosed AITL were retrospectively enrolled in the present study. For all of them, EBV serology data were available. Results: Of 9 patients, 7 (77.7%) had a triple-positive serology (VCA-IgG, VCA-IgM, EBNA-IgG ) for EBV. These patients were characterized by bone marrow involvement, high incidence of thrombocytopenia, and poor prognosis according to Revised International Prognostic Index and Prognostic Index for Angioimmunoblastic T-cell Lymphoma scores. Conclusion: Assessment of both viremia and serology for EBV could be useful in patients with clinical and laboratory data suggesting lymphoma diagnosis; furthermore, although our data need to be validated in a larger cohort of patients, triple positivity for EBV serology might help to direct the diagnosis toward AITL.

Primary pancreatic lymphoma (PPL) is a rare disease representing 0.1% of malignant lymphomas, which lacks well-defined diagnostic and therapeutic protocols. Objectives To describe PPL clinical, diagnostic and histological characteristics, together with therapy and outcome, in a relatively large series of patients. Methods The study includes 39 PPL patients, aged ≥15 years, observed from January 2005 to December 2018, in 8 Italian Institutions. Results The main symptoms were abdominal pain (58%) and jaundice (47%). Lactate dehydrogenase serum levels were elevated in 43% of patients. Histological specimens were mostly obtained by percutaneous (41%) or endoscopic (36%) biopsy, with diffuse large B-cell lymphoma being the most frequent (69%) histological diagnosis. Chemotherapy was administered alone in 65% of patients, with radiotherapy in 17%, or after surgery in 9%. The 2-year overall survival (OS) was 62%, the 2-year progression-free survival (PFS) 44%. Debulking surgery (with or without chemotherapy) was associated with a significant worse OS. Three (9.4%) of 32 high-grade patients experienced a central nervous system (CNS) relapse. Conclusions PPL is rare, often high-grade, with symptoms and localization similar to other pancreatic malignancies. Biopsy should be the preferred diagnostic method. High-grade PPL should undergo CNS prophylaxis.

Background Data on the reliability of the Ki-67 index and grading calculations from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of pancreatic neuroendocrine tumors (PanNETs) are controversial. We aimed to assess the accuracy of these data compared with histology. Methods Cytological analysis from EUS-FNA in patients with suspected PanNETs (n = 110) were compared with resection samples at a single institution. A minimum of 2000 cells were considered to be adequate for grading. Correlation and agreement between cytology and histology in grading and Ki-67 values, respectively, were investigated. Secondary outcomes included the diagnostic performance of EUS-FNA. Results EUS-FNA samples were adequate for PanNET diagnosis and PanNET grading in 98/110 (89.1 %) and 77/110 (70.0 %) patients, respectively; thus, 77 samples were adequate for comparing cytology vs. histology. There were 67 (62.0 %), 40 (36.4 %), and 1 (0.9 %) patients with a final diagnosis of G1, G2, and G3 tumors, respectively. EUS-FNA grading was concordant with surgical pathology in 81.8 % of patients; under- and overgrading occurred in 15.6 % and 2.6 %, respectively. The overall level of agreement for grading was moderate (Cohen’s κ = 0.59, 95 % confidence interval [CI] 0.34 – 0.78). Spearman’s rho for Ki-67 in tumors ≤ 20 mm and > 20 mm was strong and moderate, respectively (rho = 0.68, 95 %CI 0.47 – 0.83; rho = 0.59, 95 %CI 0.35 – 0.75). The Bland – Altman plot showed that the Ki-67 values were comparable and reproducible between the two measurements. Conclusions Although they were not available for a significant number of patients, grading and Ki-67 values from cytology correlated with histology moderately to strongly.

International audience; AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

Many studies have examined the diagnostic concordance of whole slide imaging (WSI) and light microscopy (LM) for surgical pathology. In cytopathology, WSI use has been more limited, mainly because of technical issues. The aim of this study was to review the literature and determine the overall diagnostic concordance of WSI and LM in cytopathology. A systematic search of PubMed, Scopus, and the Cochrane Library was performed, with data extracted from the included articles. A quality assessment of studies was performed with a modified Quality Assessment of Diagnostic Accuracy Studies 2 tool. The primary outcome was concordance for the diagnoses rendered by WSI and LM as shown by the concordance rate with the original diagnosis, intra-observer and interobserver concordance with the κ coefficient, or a percentage. Secondary outcomes included the time taken to reach a diagnosis and the quality and perception of WSI. A descriptive survey was provided. Among 1867 publications, a total of 19 studies (1%) were included. Overall, the concordance between WSI and the original diagnosis was 84.1%, the intra-observer concordance between WSI and LM was 92.5% with a κ coefficient of 0.66, and the interobserver κ coefficient was 0.69. The time to reach a diagnosis was longer with WSI in all studies. The quality of WSI was good, but diagnostic confidence and cytologist preference were higher for LM. In conclusion, the concordance of WSI with LM is acceptable and in line with systematic reviews in surgical pathology. However, the time required for scanning and technical issues represent barriers to complete adoption. It is foreseeable that technical advances and rigorous validation study design will help to improve the diagnostic concordance of WSI with LM in cytopathology.

Background and Objectives: Despite rarely, serous cystic adenoma (SCA) can assume a pseudo-solid aspect mimicking other pancreatic neoplasm as neuroendocrine tumor. EUS-FNA cytology has low diagnostic accuracy due to the scant cellularity of the collected samples. Histological diagnosis is usually made after resection. Recently, end-cutting needles for EUS-fine-needle biopsy (EUS-FNB), which obtain tissue cores by penetrating the lesions, have been developed. We aimed to assess the capability of EUS-FNB with SharkCore™ needles in the preoperative diagnosis of serous cystic adenoma pseudo-solid-appearing on imaging (Sa-SCA). Materials and Methods: Between January 2016 and January 2018, data from consecutive adult patients, who were referred for EUS-FNB of a solid pancreatic lesion and were diagnosed with having SCA, were retrieved from a single-center institutional database. Results: Two patients were excluded because of microcystic aspect at EUS. Histological diagnosis of SCA was made by EUS-FNB in the remaining 7 patients (5 females; mean age of 62.5 years). Lesions (mean size of 19.8 mm) were hypervascular on cross-sectional imaging, slightly hyperdense magnetic resonance imaging with T2-weighted images can, and negative at 68Ga-somatostatin receptor positron emission tomography and 18fluoro-deoxyglucose positron emission tomography. EUS-FNB samples were judged adequate for a definitive diagnosis in all cases, achieving specimens suitable for histological evaluation and several ancillary stains. Histochemical positivity for periodic acid-Schiff (PAS) and PAS with diastase digestion was observed in 7/7 cases. Immunohistochemical positivity for α-inhibin (7/7), GLUT1 (6/6), MUC6 (5/5), and negativity for synaptophysin (7/7) and chromogranin A (2/2) favored SCA diagnosis. Conclusions: In the case of preoperative workup suspected for Sa-SCA, a “forward acquiring” needle could improve the rate of preoperative histological diagnosis.

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.

EUS-guided through-the-needle biopsy (TTNB) sampling has been reported to improve diagnostic yield compared with cytology for the evaluation of pancreatic cystic lesions (PCLs). The number of macroscopically visible tissue samples needed to reach an adequate diagnosis is still unknown.This is a retrospective, single-center study on consecutive patients with PCLs with risk features (cyst3 cm, thickened wall, cyst growth during follow-up, and mural nodules) who underwent TTNB sampling. The capability of differentiating mucinous versus nonmucinous cysts, ability to obtain a cyst-lining epithelium, definition of the grade of dysplasia, and specific diagnosis of cyst histotype were evaluated for 1, 2, or 3 TTNB macroscopically visible specimens.Sixty-one patients were evaluated. A 100% histologic adequacy was reached by 2 samples (P = .05 versus 1). Compared with cytology, 1 TTNB specimen improved the possibility of defining cyst histotype (P .0001), whereas 2 specimens increased all 4 diagnostic categories (P .003). Two specimens also increased diagnostic yield compared with 1 sample (P .085). The collection of a third sample did not improve the value of any diagnostic categories. A specific diagnosis was reached in 74% of patients with 2 histologic samples. The diagnostic reliability of TTNB sampling compared with surgical histology was 90%, with a 22.9% rate of adverse events.Two TTNB macroscopically visible specimens reached 100% histologic adequacy and a specific diagnosis in 74% of patients. The collection of a third specimen did not add any additional information and should be avoided to possibly decrease the risk of adverse events.

The association of systemic mastocytosis with another hematologic neoplasia of myeloid or lymphoid origin is recognized as an advanced subvariant of mastocytosis. Here, we report the association of indolent or smoldering systemic mastocytosis with three cases of myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis, a recently recognized disease characterized by SF3B1 mutations. The hierarchical pattern of KIT, SF3B1, JAK2, and additional mutations was studied in whole and fractionated subpopulations of peripheral blood cells and whole bone marrow. In two cases, we could demonstrate a multilineage D816V KIT mutation, involving all myeloid lineages in one patient and also the lymphoid series in the other. Two patients displaying both SF3B1 and V617F JAK2 mutations had a very poor prognosis. Another patient bearing SF3B1, but not V617F JAK2 mutation, had a favorable response to erythropoietin treatment and long survival.

// Andrea Mafficini 1,* , Michele Simbolo 1,* , Alice Parisi 2 , Borislav Rusev 1,2 , Claudio Luchini 1,2 , Ivana Cataldo 1 , Elena Piazzola 2 , Nicola Sperandio 1 , Giona Turri 2 , Massimo Franchi 3 , Giampaolo Tortora 4 , Chiara Bovo 5 , Rita T. Lawlor 1,2 and Aldo Scarpa 1,2 1 ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy 2 Department of Pathology & Diagnostics, University and Hospital Trust of Verona, Verona, Italy 3 Department of Gynecology, University and Hospital Trust of Verona, Verona, Italy 4 Comprehensive Cancer Centre, University and Hospital Trust of Verona, Verona, Italy 5 Board of Directors, University and Hospital Trust of Verona, Verona, Italy * Shared first authors Correspondence to: Rita T. Lawlor , email: // Keywords : BRCA1-BRCA2 , ovarian carcinoma, next generation sequencing, PARP inhibitor, olaparib Received : November 10, 2015 Accepted : December 29, 2015 Published : January 07, 2016 Abstract BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2 . All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.

The aim of this study was to compare the performance of EUS-guided fine-needle biopsy using fork-tip or side-fenestrated needles in patients with solid pancreatic lesions.A randomized controlled study was conducted in a single academic center on patients who underwent sampling with fork-tip or side-fenestrated 22-gauge or 25-gauge needles. Three passes were performed, each independently evaluated by a blinded pathologist and by endosonographers for macroscopic on-site evaluation (MOSE). The primary outcome was histologic yield; secondary aims were safety, diagnostic yield, sample quality, number of needle passes required to establish a diagnosis, and reliability of MOSE.One hundred ninety-two patients were enrolled. Both 22-gauge and 25-gauge fork-tip needles retrieved significantly higher rates of histologic samples than side-fenestrated needles (P .013). Safety and diagnostic accuracy were comparable in the 2 arms, whereas sample quality (tissue integrity and blood contamination) was significantly better in the fork-tip group (P .0001). The median number of diagnostic passes was lower using fork-tip needles (P = .054). The agreement between MOSE and pathologic evaluation was almost perfect in the fork-tip group and fair in the side-fenestrated group.Both needles showed equivalent safety and diagnostic accuracy. However, fork-tip needles provided a higher rate of extremely good-quality histologic samples and required fewer needle passes to reach a diagnosis. MOSE is a highly reliable tool when fork-tip needles are used compared with side-fenestrated needles. (Clinical trial registration number: NCT03622229.).

AMP-activated protein kinase (AMPK) is a central energy sensor and master regulator of energy homeostasis. AMPK not only elicits acute metabolic responses, but also promotes metabolic reprogramming and adaptions in the long-term through regulation of specific transcription factors/co-activators. We performed a whole-genome transcriptome profiling in wild-type and AMPK-deficient mouse embryonic fibroblasts (MEF) and primary hepatocytes that had been treated with two distinct classes of small-molecule AMPK activators, namely 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or 991. This led to the identification of distinct compound-dependent gene expression signatures and to the discovery of several AMPK-regulated genes, including Flcn encoding a tumor suppressor and nutrient sensor folliculin (FLCN). Gene set enrichment and pathway analyses identified the lysosomal pathway and the associated transcription factor EB (TFEB) as key transcriptional mediator responsible for AMPK-dependent gene expression changes. AMPK-induced Flcn expression was abolished in TFEB/TFE3 double knockout MEF and the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. Mechanistically, we have found that AMPK promotes the dephosphorylation and nuclear localization of TFEB independently of mTOR activity. Collectively, we identified the AMPK-TFEB-FLCN axis as a potential key regulator for cellular and metabolic homeostasis. Moreover, data from zebrafish with physiologically and pharmacologically activated AMPK supports the existence of the AMPK-TFEB-FLCN cascade in vivo.

Fish species display huge differences in physical activity ranging from lethargy to migration of thousands of miles, making them an interesting model to identify determinants of physical fitness. Here, we show a remarkable plasticity of zebrafish in response to exercise and induction of PGC1α (encoded byPPARGC1A), a dominant regulator of mitochondrial biogenesis. Forced expression of humanPPARGC1Ainduces mitochondrial biogenesis, an exercise-like gene expression signature, and physical fitness comparable to wild-type animals trained in counter-current swim tunnels. Quantifying transcriptional and proteomic changes in response to exercise or PGC1α, we identify conserved ‘exercise’ adaptations, including a stoichiometric induction of the electron transport chain (ETC) that re-organizes into respiratory supercomplexes in both conditions. We further show that ndufa4/ndufa4l, previously assigned to complex I, associates to free and supramolecular complex IVin vivo. Thus, zebrafish is a useful and experimentally tractable vertebrate model to study exercise biology, including ETC expression and assembly.HIGHLIGHTSPGC1α reprograms zebrafish skeletal muscle to a ‘red fiber’ phenotype and increases exercise performanceZebrafish show a high molecular plasticity in response to PGC1α and exerciseSWATH-MS proteomics show a stoichiometric induction of the electron transport chain that organizes as supercomplexes in response to PGC1α and exercisendufa4/ndufa4l associate to free and supramolecular complex IVin vivo

In muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression., The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues.

Background Early detection of small solid pancreatic lesions is increasingly common. To date, few and contradictory data have been published about the relationship between lesion size and endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) diagnostic yield. The aim of this study was to assess the relation between the size of solid pancreatic lesions and the diagnostic yield of EUS-FNA using a 25-gauge needle in a center without available rapid on-site evaluation. Methods In the retrospective cohort study, we selected patients who underwent EUS-FNA for solid pancreatic lesions with a 25-gauge needle from October 2014 to October 2015. Patients were divided into three groups (≤15 mm, 16–25 mm and >25 mm), and the outcomes were compared. Results We analyzed 163 patients. Overall adequacy, sensitivity, specificity and accuracy were 85.2%, 81.8%, 93.7%, and 80.4%, respectively. When stratified by size, the sensitivity and accuracy correlated with size (P = 0.016 and P = 0.042, respectively). Multivariate analysis showed that lesion size was the only independent factor (P = 0.019, OR = 4.76) affecting accuracy. The role of size as an independent factor affecting accuracy was confirmed in a separate multivariate analysis, where size was included in the model as a covariate (P = 0.018, OR = 1.08). Conclusion Our study demonstrates that, in the absence of rapid on-site evaluation, mass size affects the accuracy of EUS-FNA of solid pancreatic lesions.

Key Clinical Message Patients with advanced variants of Systemic Mastocytosis may develop destructive bone lesions when massive mast cell (MC) infiltrates are present. Finding of large osteolyses in indolent systemic mastocytosis, typically characterized by low MC burden, should prompt investigations for an alternative explanation.

International audience; Tight regulation of cell proliferation and differentiation is required to ensure proper growth during development and post-natal life. The source and nature of signals regulating cell proliferation are not well identified in vivo. We investigated the specific pattern of proliferating cells in mouse limbs, using the Fluorescent ubiquitynation-based cell-cycle indicator (Fucci) system, which allowed the visualization of the G1, G1/S transition and S/G2/M phases of the cell cycle in red, yellow or green fluorescent colors, respectively. We also used the retroviral RCAS system to express a Fucci cassette in chick embryos. We performed a comprehensive analysis of the cell cycle state of myogenic cells in fetal limb muscles, adult myoblast primary cultures and isolated muscle fiber cultures using the Fucci transgenic mice. We found that myonuclei of terminally differentiated muscle fibers displayed Fucci red fluorescence during mouse and chick fetal development, in adult isolated muscle fiber (ex vivo) and adult myoblast (in vitro) mouse cultures. This indicated that myonuclei exited from the cell cycle in the G1 phase and are maintained in a blocked G1-like state. We also found that cycling muscle progenitors and myoblasts in G1 phase were not completely covered by the Fucci system. During mouse fetal myogenesis, Pax7+ cells labeled with the Fucci system were observed mostly in S/G2/M phases. Proliferating cells in S/G2/M phases displayed a specific pattern in mouse fetal limbs, delineating individualized muscles. In addition, we observed more Pax7+ cells in S/G2/M phases at muscle tips, compared to the middle of muscles. These results highlight a specific spatial regionalization of cycling cells at the muscle borders and muscle-tendon interface during fetal development.

Mitochondrial abundance and function are tightly controlled during metabolic adaptation, but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5′ untranslated region of its gene (PPARGC1A). We find that uORF–mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism, and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function.

Background: Hepatoid carcinomas (HCs) are extrahepatic neoplasms exhibiting features of hepatocellular tumors in terms of morphology and immunohistochemistry. They have been described in several organs, most notably in the stomach and ovary. They can present in pure forms or in association with other morphological aspects, such as endocrine tumors or ductal adenocarcinomas. The aim of this review is to describe aspects of hepatoid adenocarcinoma of the pancreas with regard to epidemiology, diagnosis, and treatment. Methods: The PubMed database was searched for publications addressing hepatoid adenocarcinoma of the pancreas. We have searched for articles including the following keywords: ‘pancreatic hepatoid carcinoma', ‘ectopic liver cancer' and ‘rare pancreas neoplasm' published to date. As references, we used case reports and review articles. Results: Pancreatic forms of HCs are extremely uncommon: only 22 cases have been reported. Conclusions: The possibility of an HC of the pancreas should be considered in the differential diagnosis of an uncommon pathological mass of the pancreas. Treatment seems to be related to the association with other neoplasms, tumor extension at the time of diagnosis and the possibility to perform a radical resection. The common embryologic origin of the pancreas and liver, together with peculiar environmental factors, may explain the development of pancreatic HCs.

Skeletal muscle regeneration relies on a pool of resident muscle stem cells called satellite cells (MuSCs). Following injury-induced destruction of the myofibers, quiescent MuSCs are activated and generate transient amplifying progenitors (myoblasts) that will fuse to form new myofibers. Here, we focus on the canonical Wnt signaling pathway and find that either conditional β-catenin disruption or activation in adult MuSCs results in perturbation of muscle regeneration. Using both in vivo and in vitro approaches, we observed that myoblasts lacking β-catenin show delayed differentiation, whereas myoblasts with constitutively active β-catenin undergo precocious growth arrest and differentiation. Transcriptome analysis further demonstrated that Wnt/β-catenin signaling interacts with multiple pathways and, more specifically, TGF-β signaling. Indeed, exogenous TGF-β2 stimulation restores the regenerative potential of muscles with targeted β-catenin disruption in MuSCs. We conclude that a precise level of β-catenin activity is essential for regulating the amplification and differentiation of MuSC descendants during adult myogenesis. Using muscle stem cell-specific β-catenin gain-of-function and loss-of-function mutations, Rudolf et al. demonstrate that muscle progenitor cells (MPCs) require the canonical Wnt/β-catenin pathway during skeletal muscle regeneration. In vivo and in vitro, MPCs differentiate less efficiently when β-catenin is absent, whereas conditional activation of β-catenin pushes MPCs toward precocious differentiation.

Background: Kaposiform Hemangioendothelioma (KHE) is a rare vascular tumour of the infancy and of the first decade of life. It is locally aggressive and potentially life threatening when associated to consumptive coagulopathy, known as Kasabach-Merritt syndrome (KMS). No consensus or guideline for the therapy has been reached because of the lack of prospective trials and the different standard care suggestions are based on retrospective case series. Case report: We report the case of a 9-month-old male with KHE and KMS in which the initial response, obtained with prednisone and vincristine, was subsequently consolidated and strengthened by long-term treatment with sirolimus, an mTOR inhibitor. A summary of the published data is presented as well. Conclusions: The inhibition of mTOR pathway represents the most important therapeutic innovation introduced in the last few years for KHE. Our case shows the effectiveness and good tolerance of long-term therapy with sirolimus. Keywords: Kaposiform Hemangioendothelioma, Kasabach-Merrit syndrome, sirolimus, prednisone, vincristine

Mantle cell lymphoma (MCL) is an incurable hematologic malignancy whose pathogenesis is only partly understood. The aim of the present study was to define a "core phosphoproteome" in MCL cell lines that is representative of primary MCL in order to improve knowledge of the signal transduction pathways involved in its tumorigenesis. We have analyzed phosphorylated proteins in several MCL cell lines by immobilized metal affinity chromatography and separation by 2-D PAGE, followed by RP-HPLC coupled with MS/MS identification. These data were correlated with information on copy number gains obtained by SNP-chip analysis. Several of the proteins identified could be linked to a specific signal transduction pathway, and have been recently recognized as important players in MCL pathogenesis, such as nuclear factor-kappaB (NF-kappaB) and phosphoinositide-3 kinase-mammalian target of rapamycin (PI3K-mTOR). However, our data also implicate a number of novel proteins and pathways in the pathobiology of MCL, one of which is mitochondrial signaling. A second-level analysis identified MAPK1, CK2, CK1, PKCzeta, and PKCepsilon as candidate upstream molecules. Our study provides new insights in MCL pathogenesis and helps to form the basis for testing new target-specific therapeutics.

OBJECTIVE To assess the short-term outcome in patients with high-risk prostate cancer treated by transrectal high-intensity focused ultrasound (HIFU). PATIENTS AND METHODS From April 2003 to November 2004, 30 patients with high-risk prostate cancer were enrolled in this prospective study; all had transurethral resection of the prostate before transrectal HIFU treatment, using the Ablatherm device (EDAP, Lyon, France) during the same session, associated with hormonal therapy with luteinizing hormone-releasing hormone analogues. After the procedure, all the patients were evaluated every 3 months by physical examination, prostate-specific antigen (PSA) assay and a continence questionnaire. The follow-up schedule also included a transperineal prostate biopsy 6 months after the treatment. All the patients had a minimum follow-up of 12 months. RESULTS The HIFU treatment took a median (interquartile range, IQR) of 140 (100-160) min. No complications were reported during treatment. The mean (IQR) hospitalization was 2.2 (1-4) days, and the suprapubic drainage tube was removed after 12 (7-18) days. The complications after treatment were: urinary tract infections in five patients (16%), stenosis of the intraprostatic and membranous urethra in three (10%), and secondary infravesical obstruction in four (13%). At 12 months after the procedure, 28 patients (93%) were continent. Seven of the 30 men (23%) had a positive prostate biopsy. At the 1-year follow-up only three of the 30 patients with high-risk prostate cancer had a PSA level of >0.3 ng/mL. CONCLUSIONS HIFU is a modern, minimally invasive therapy for prostate cancer, often used in selected patients with localized disease. The present results show that HIFU was also feasible in patients with high-risk prostate cancer. The low complication rates and favourable functional outcome support the planning of further larger studies in such patients. The oncological efficacy of HIFU should be assessed in further studies with a longer follow-up.

Background Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. Methods PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. Results PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. Conclusion Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes. Electronic supplementary material The online version of this article (doi:10.1007/s13402-011-0019-7) contains supplementary material, which is available to authorized users.

Summary Aims High intensity focused ultrasound (HIFU) is an emerging alternative for the treatment of prostate adenocarcinoma. Alpha-methylacyl-CoA racemase (AMACR) has been shown to be a sensitive immunomarker for prostate cancer, however, there is no information available concerning its utility and that of other immunomarkers for the detection of malignancy after HIFU therapy. Methods AMACR expression was examined in 11 cases of prostatic carcinomatreated byHIFU, with histological evidence of residual carcinoma. In seven cases tumour was examined from thin core biopsies and in four cases fromtissue fragments obtained by transurethral resection of prostate (TURP). In addition to AMACR, immunostaining was also undertaken for p63, cytokeratin 34βE12, cytokeratin 5, cytokeratin 8-18, prostate specific alkaline phosphatase (PSAP), prostate specific antigen (PSA), chromogranin and CD56. Results In two of the cases foci of tumour were cut out in serial sections. AMACR was expressed in eight of nine evaluable cases (4/5 biopsies and 4/4 TURP specimens). Cytokeratin 8-18 and PSAP were positive in all cases, whereas PSA was positive in five of nine cases. Cytokeratin 34βE12, cytokeratin 5, and p63 marked the basal layer in normal prostatic glands, but were negative in neoplastic glands. In four cases we found tumour cells with positive staining for CD56 and chromogranin. Conclusions A panel with positive markers for AMACR, and negative markers for p63/cytokeratin 5/cytokeratin 34βE12 confirms the neoplastic nature of the residual glands on biopsies or TURP fragments sampled after HIFU therapy.

The clinical significance and management (surgical excision vs. follow-up) of the patients with the diagnosis of flat epithelial atypia (FEA) on core needle biopsy (CNB) are actually under discussion. Using standardized criteria and precise terminology, we analyzed retrospectively our CNB diagnosis of FEA, dividing patients with pure FEA as the most advanced pathologic lesion from patients with FEA associated to atypical ductal hyperplasia (FEA+ADH). Both the categories were correlated with radiologic data and findings on subsequent surgery. We evaluated 875 core needle biopsies (11-gauge stereotactic vacuum-assisted procedure), performed over a 5-year period. A CNB diagnosis of pure FEA was made in 33/875 (3.7%) cases; in other 11 (1.2%) cases we observed the coexistence of FEA and ADH. Subsequent surgical excisions were available in 20/33 pure FEA and in 10/11 FEA+ADH: of the 20 patients with pure FEA on CNB, none had either ductal carcinoma in situ or invasive carcinoma in their excisional biopsy, whereas 3/10 (30%) FEA + ADH on CNB showed, at subsequent surgery, more advanced lesions (2 ductal carcinoma in situ, 1 invasive carcinoma). Our results suggest that patients with an 11-gauge vacuum-assisted CNB diagnosis of pure FEA (especially if related to a small radiologic target, completely or almost completely removed by the needle biopsy procedure) could be spared surgical excision and managed with close radiologic follow-up.