Molecular investigation of ALK-rearranged epithelioid fibrous histiocytomas identifies CLTC as a novel fusion partner and evidence of fusion-independent transcription activation.

Epithelioid fibrous histiocytoma (EFH) is a rare cutaneous neoplasm, which is characterized by the presence of rearrangements involving the ALK gene. Although EFH was long considered a variant of fibrous histiocytoma, the identification of its unique genetic signature confirmed that it represents a distinct entity. The aim of the present study was to examine a cohort of ALK-immunoreactive EFH cases to further characterize gene fusion partners. Next generation sequencing detected ALK fusions in 11 EFH cases identified in the pathology archives of two different institutions. The most common fusion partner was DCTN1 (N = 4) followed by CLTC (N = 2) and VCL (N = 2), while the remaining cases harbored fusions involving SPECC1L, PPFIBP1, and PRKAR1A. In one case no fusion was detected by NGS and FISH despite suitable sample quality. Notably, IHC demonstrated positive ALK expression and the level of aligned ALK reads was comparable to the fusion-positive cases. To the best of our knowledge, this is the first report of CLTC as a fusion partner in EFH. The two CLTC rearranged cases in our cohort also represent the first two EFH cases in the literature that involve exon 19 of ALK, instead of exon 20. These findings underscore the remarkable plasticity of ALK as an oncogenic driver and further expand the list of its potential fusion partners in EFH. Lastly this is also the first report of ALK-immunoreactive EFH with no underlying fusion suggesting a fusion independent transcription mechanism as seen in other tumors.

EWSR1-PATZ1 fusion renal cell carcinoma: a recurrent gene fusion characterizing thyroid-like follicular renal cell carcinoma.

Thyroid-like follicular renal cell carcinoma is an uncommon kidney tumor with no distinct molecular alteration described to date. This cohort of eight women with mean and median ages of 45 and 46 years, respectively (range 19-65 years), had unencapsulated, well-circumscribed tumors composed of tightly packed anastomosing follicle-like cysts filled with eosinophilic colloid-like material and lined by cuboidal cells with high nuclear to cytoplasmic ratios, oval to elongated nuclei with perpendicular arrangement toward the lumens, and prominent nuclear overlapping. The stroma between these was minimal with the exception of two tumors. Calcifications and necrosis were absent. Immunohistochemically, the tumors were positive for KRT19 (7/7), PAX8 (5/5), cyclin D1 (6/6), KRT7 (5/7), and AMACR (1/5; focal, weak), and were negative for WT1, TTF1 (transcription termination factor-1), and thyroglobulin. In three of three tumors tested molecularly, EWSR1-PATZ1 fusion was identified by RNA sequencing and confirmed by RT-PCR and Sanger sequencing. Over a follow-up period of 1-7 years, no evidence of recurrence or metastasis has been detected. The EWSR1-PATZ1 fusion has been recognized as a recurrent alteration in a subset of round to spindle cell sarcomas with EWSR1-non-ETS fusions (EWSR1-PATZ1 sarcoma) and in several central nervous system tumors. The finding of an EWSR1-PATZ1 fusion in all three of the thyroid-like follicular renal cell carcinomas for which sufficient tissue was available for genomic profiling provides the first distinct molecular abnormality in thyroid-like follicular renal cell carcinomas, supporting its designation as a distinct diagnostic entity.

Second Report of PDE10A-BRAF Fusion in Pediatric Spindle Cell Sarcoma With Infantile Fibrosarcoma-Like Morphology Suggesting PDE10A-BRAF Fusion Is a Recurrent Event.

Infantile/congenital fibrosarcoma (IFS) is the most common soft tissue tumor in children less than one year of age. The most common anatomic site of IFS is in the extremities or trunk, and rarely in the abdomen or retroperitoneum. Approximately 70-90% of cases are characterized by a distinct t(12;15)(p13;q25) translocation resulting in an ETV6-NTRK3 gene fusion. As such, TRK inhibitors are considered frontline therapy in TRK-fusion positive IFS. The ETV6-NTRK3 fusion is also detected in congenital mesoblastic nephroma (CMN) and less frequently in myeloid leukemias, secretory breast carcinoma, and mammary-type secretory carcinoma of the skin and salivary glands. Infrequently, cases of tumors with IFS-like morphology without the characteristic ETV6-NTRK3 gene fusion have been identified. Herein, an ETV6-NTRK3 fusion negative spindle cell sarcoma with IFS-like morphology subjected to genomic profiling revealed a PDE10A-BRAF fusion, a fusion event that has been detected previously in an isolated case of undifferentiated infantile sarcoma.

A novel CLTC-FOSB gene fusion in pseudomyogenic hemangioendothelioma of bone.

Pseudomyogenic hemangioendothelioma, an uncommon mesenchymal neoplasm composed of plump spindled and/or epithelioid endothelial cells, may present multicentrically and tends to locally recur but rarely metastasizes. Morphologic resemblance to epithelioid sarcoma and other spindle cell neoplasms may result in diagnostic confusion. Molecular characterization of pseudomyogenic hemangioendothelioma has revealed these neoplasms often harbor a rearrangement of the FOSB gene with SERPINE1 or ACTB as recurrent fusion gene partners. Herein, a case of a fibular pseudomyogenic hemangioendothelioma with minimal extension into the adjacent soft tissue arising in a 17 year-old male is presented. The neoplasm exhibited sheets of epithelioid cells with abundant eosinophilic cytoplasm and variably eccentric nuclei. RNA sequencing revealed a novel CLTC-FOSB fusion transcript that was subsequently confirmed by direct sequencing of reverse transcription-polymerase chain reaction products demonstrating an in-frame fusion between exon 17 of the clathrin heavy chain (CLTC) gene and exon 2 of the FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) gene. CLTC-FOSB fusion has not been described in a neoplasm before.

Hypertriglyceridemia-induced acute pancreatitis: A prospective, multicenter, international cohort analysis of 716 acute pancreatitis cases.

BACKGROUND: Hypertriglyceridemia is the third most common cause of acute pancreatitis (AP). It has been shown that hypertriglyceridemia aggravates the severity and related complications of AP; however, detailed analyses of large cohorts are contradictory. Our aim was to investigate the dose-dependent effect of hypertriglyceridemia on AP. METHODS: AP patients over 18 years old who underwent triglyceride measurement within the initial three days were included into our cohort analysis from a prospective international, multicenter AP registry operated by the Hungarian Pancreatic Study Group. Data on 716 AP cases were analyzed. Six groups were created based on the highest triglyceride level (<1.7 mmol/l, 1.7-2.19 mmol/l, 2.2-5.59 mmol/l, 5.6-11.29 mmol/l, 11.3-22.59 mmol/l, ≥22.6 mmol/l). RESULTS: Hypertriglyceridemia (≥1.7 mmol/l) presented in 30.6% of the patients and was significantly and dose-dependently associated with younger age and male gender. In 7.7% of AP cases, hypertriglyceridemia was considered as a causative etiological factor (≥11.3 mmol/l); however, 43.6% of these cases were associated with other etiologies (alcohol and biliary). Hypertriglyceridemia was significantly and dose-dependently related to obesity and diabetes. The rates of local complications and organ failure and maximum CRP level were significantly and dose-dependently raised by hypertriglyceridemia. Triglyceride above 11.3 mmol/l was linked to a significantly higher incidence of moderately severe AP and longer hospital stay, whereas triglyceride over 22.6 mmol/l was significantly associated with severe AP as well. CONCLUSION: Hypertriglyceridemia dose-dependently aggravates the severity and related complications of AP. Diagnostic workup for hypertriglyceridemia requires better awareness regardless of the etiology of AP.

Superficial malignant ossifying fibromyxoid tumors harboring the rare and recently described ZC3H7B-BCOR and PHF1-TFE3 fusions.

Ossifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain differentiation and intermediate biologic potential. Up to 85% of OFMTs, including benign, atypical, and malignant forms, harbor fusion genes. Most commonly, the PHF1 gene localized to 6p21 is fused with EP400, but other fusion partners, such as MEAF6, EPC1, and JAZF1 have also been described. Herein, we present two rare cases of superficial OFMTs with ZC3H7B-BCOR and the very recently described PHF1-TFE3 fusions. The latter also exhibited moderate to strong diffuse immunoreactivity for TFE3. Reciprocally, this finding expands the entities with TFE3 rearrangements. Accumulation of additional data is necessary to determine if OFMTs harboring these rare fusions feature any reproducible clinicopathologic findings or carry prognostic and/or predictive implications.

Epithelioid Fibrous Histiocytoma With Dot-Like Perinuclear ALK Expression and PRKAR2A-ALK Fusion.

Epithelioid fibrous histiocytoma (EFH) is a rare, benign, cutaneous neoplasm. This fibrohistiocytic tumor was once believed to be a variant of fibrous histiocytoma, but EFH is now known to be a distinct entity based on the presence of ALK gene rearrangements in most cases. The pattern of immunohistochemical expression of ALK in EFH in the literature thus far describes both granular cytoplasmic staining and nuclear staining. We present a case of EFH with dot-like Golgi pattern perinuclear ALK expression, a previously undescribed staining pattern. We surmised this unique staining pattern could be due to a novel fusion partner, and using FISH, we confirmed a rearrangement of the ALK (2p23) locus. Further investigation with whole transcriptome sequencing led to the discovery of PRKAR2A-ALK fusion, and the function of this fusion partner reflects a Golgi-predominant localization of the protein. Attention to the distinct immunohistochemical pattern of ALK expression may provide clues to the function of the fusion partner.

A Multicenter, International Cohort Analysis of 1435 Cases to Support Clinical Trial Design in Acute Pancreatitis.

BACKGROUND: C-reactive protein level (CRP) and white blood cell count (WBC) have been variably used in clinical trials on acute pancreatitis (AP). We assessed their potential role. METHODS: First, we investigated studies which have used CRP or WBC, to describe their current role in trials on AP. Second, we extracted the data of 1435 episodes of AP from our registry. CRP and WBC on admission, within 24 h from the onset of pain and their highest values were analyzed. Descriptive statistical tools as Kruskal-Wallis, Mann-Whitney U, Levene's F tests, Receiver Operating Characteristic (ROC) curve analysis and AUC (Area Under the Curve) with 95% confidence interval (CI) were performed. RESULTS: Our literature review showed extreme variability of CRP used as an inclusion criterion or as a primary outcome or both in past and current trials on AP. In our cohort, CRP levels on admission poorly predicted mortality and severe cases of AP; AUC: 0.669 (CI:0.569-0.770); AUC:0.681 (CI: 0.601-0.761), respectively. CRP levels measured within 24 h from the onset of pain failed to predict mortality or severity; AUC: 0.741 (CI:0.627-0.854); AUC:0.690 (CI:0.586-0.793), respectively. The highest CRP during hospitalization had equally poor predictive accuracy for mortality and severity AUC:0.656 (CI:0.544-0.768); AUC:0.705 (CI:0.640-0.769) respectively. CRP within 24 h from the onset of pain used as an inclusion criterion markedly increased the combined event rate of mortality and severe AP (13% for CRP > 25 mg/l and 28% for CRP > 200 mg/l). CONCLUSION: CRP within 24 h from the onset of pain as an inclusion criterion elevates event rates and reduces the number of patients required in trials on AP.

Clinical, pathological, and genomic features of EWSR1-PATZ1 fusion sarcoma.

Molecular diagnostics of sarcoma subtypes commonly involve the identification of characteristic oncogenic fusions. EWSR1-PATZ1 is a rare fusion partnering in sarcoma, with few cases reported in the literature. In the current study, a series of 11 cases of EWSR1-PATZ1 fusion positive malignancies are described. EWSR1-PATZ1-related sarcomas occur across a wide age range and have a strong predilection for chest wall primary site. Secondary driver mutations in cell-cycle genes, and in particular CDKN2A (71%), are common in EWSR1-PATZ1 sarcomas in this series. In a subset of cases, an extended clinical and histopathological review was performed, as was confirmation and characterization of the fusion breakpoint revealing a novel intronic pseudoexon sequence insertion. Unified by a shared gene fusion, EWSR1-PATZ1 sarcomas otherwise appear to exhibit divergent morphology, a polyphenotypic immunoprofile, and variable clinical behavior posing challenges for precise classification.

Antibiotic therapy in acute pancreatitis: From global overuse to evidence based recommendations.

BACKGROUND: Unwarranted administration of antibiotics in acute pancreatitis presents a global challenge. The clinical reasoning behind the misuse is poorly understood. Our aim was to investigate current clinical practices and develop recommendations that guide clinicians in prescribing antibiotic treatment in acute pancreatitis. METHODS: Four methods were used. 1) Systematic data collection was performed to summarize current evidence; 2) a retrospective questionnaire was developed to understand the current global clinical practice; 3) five years of prospectively collected data were analysed to identify the clinical parameters used by medical teams in the decision making process, and finally; 4) the UpToDate Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system was applied to provide evidence based recommendations for healthcare professionals. RESULTS: The systematic literature search revealed no consensus on the start of AB therapy in patients with no bacterial culture test. Retrospective data collection on 9728 patients from 22 countries indicated a wide range (31-82%) of antibiotic use frequency in AP. Analysis of 56 variables from 962 patients showed that clinicians initiate antibiotic therapy based on increased WBC and/or elevated CRP, lipase and amylase levels. The above mentioned four laboratory parameters showed no association with infection in the early phase of acute pancreatitis. Instead, procalcitonin levels proved to be a better biomarker of early infection. Patients with suspected infection because of fever had no benefit from antibiotic therapy. CONCLUSIONS: The authors formulated four consensus statements to urge reduction of unjustified antibiotic treatment in acute pancreatitis and to use procalcitonin rather than WBC or CRP as biomarkers to guide decision-making.

Prospective, Multicentre, Nationwide Clinical Data from 600 Cases of Acute Pancreatitis.

OBJECTIVE: The aim of this study was to analyse the clinical characteristics of acute pancreatitis (AP) in a prospectively collected, large, multicentre cohort and to validate the major recommendations in the IAP/APA evidence-based guidelines for the management of AP. DESIGN: Eighty-six different clinical parameters were collected using an electronic clinical research form designed by the Hungarian Pancreatic Study Group. PATIENTS: 600 adult patients diagnosed with AP were prospectively enrolled from 17 Hungarian centres over a two-year period from 1 January 2013. MAIN RESULTS: With respect to aetiology, biliary and alcoholic pancreatitis represented the two most common forms of AP. The prevalence of biliary AP was higher in women, whereas alcoholic AP was more common in men. Hyperlipidaemia was a risk factor for severity, lack of serum enzyme elevation posed a risk for severe AP, and lack of abdominal pain at admission demonstrated a risk for mortality. Abdominal tenderness developed in all the patients with severe AP, while lack of abdominal tenderness was a favourable sign for mortality. Importantly, lung injury at admission was associated with mortality. With regard to laboratory parameters, white blood cell count and CRP were the two most sensitive indicators for severe AP. The most common local complication was peripancreatic fluid, whereas the most common distant organ failure in severe AP was lung injury. Deviation from the recommendations in the IAP/APA evidence-based guidelines on fluid replacement, enteral nutrition and timing of interventions increased severity and mortality. CONCLUSIONS: Analysis of a large, nationwide, prospective cohort of AP cases allowed for the identification of important determinants of severity and mortality. Evidence-based guidelines should be observed rigorously to improve outcomes in AP.

Genetic analysis of the bicarbonate secreting anion exchanger SLC26A6 in chronic pancreatitis.

BACKGROUND: Pancreatic ductal HCO3(-) secretion is critically dependent on the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) and the solute-linked carrier 26 member 6 anion transporter (SLC26A6). Deterioration of HCO3(-) secretion is observed in chronic pancreatitis (CP), and CFTR mutations increase CP risk. Therefore, SLC26A6 is a reasonable candidate for a CP susceptibility gene, which has not been investigated in CP patients so far. METHODS: As a first screening cohort, 106 subjects with CP and 99 control subjects with no pancreatic disease were recruited from the Hungarian National Pancreas Registry. In 60 non-alcoholic CP cases the entire SLC26A6 coding region was sequenced. In the Hungarian cohort variants c.616G > A (p.V206M) and c.1191C > A (p.P397=) were further genotyped by restriction fragment length polymorphism analysis. In a German replication cohort all exons were sequenced in 40 non-alcoholic CP cases and variant c.616G > A (p.V206M) was further analyzed by sequencing in 321 CP cases and 171 controls. RESULTS: Sequencing of the entire coding region revealed four common variants: intronic variants c.23 + 78_110del, c.183-4C > A, c.1134 + 32C > A, and missense variant c.616G > A (p.V206M) which were found in linkage disequilibrium indicating a conserved haplotype. The distribution of the haplotype did not show a significant difference between patients and controls in the two cohorts. A synonymous variant c.1191C > A (p.P397=) and two intronic variants c.1248 + 9_20del and c.-10C > T were detected in single cases. CONCLUSION: Our data show that SLC26A6 variants do not alter the risk for the development of CP.

Pharmacologic inhibition of epigenetic modification reveals targets of aberrant promoter methylation in Ewing sarcoma.

BACKGROUND: Ewing sarcoma (ES), a highly aggressive tumor of children and young adults, is characterized most commonly by an 11;22 chromosomal translocation that fuses EWSR1 located at 22q12 with FLI1, coding for a member of the ETS family of transcription factors. Although genetic changes in ES have been extensively researched, our understanding of the role of epigenetic modifications in this neoplasm is limited. PROCEDURE: In an effort to improve our knowledge in the role of epigenetic changes in ES we evaluated the in vitro antineoplastic effect of the DNA methyltransferase inhibitor 5-Aza-deoxycytidine (5-Aza-dC) and identified epigenetically silenced genes by pharmacologic unmasking of DNA methylation coupled with genome-wide expression profiling. RESULTS: Comparisons between untreated and 5-Aza-dC treated ES cell lines (n = 5) identified 208 probe sets with at least twofold difference in expression (P ≤ 0.05). The 208 probe sets represented 145 upregulated and 31 down-regulated genes. Of the 145 genes upregulated after 5-Aza-dC treatment, four: were further characterized. ACRC, CLU, MEST, and NNAT were found to be hypermethylated and transcriptionally down-regulated in ES cell lines. Further studies revealed that ACRC, CLU, MEST, and NNAT were often hypermethylated in primary ES tumors. Transfection-mediated reexpression of ACRC, CLU, MEST, and NNAT in ES cell lines resulted in decreased growth in culture. CONCLUSIONS: This study demonstrated epigenetically modified genes in ES cell lines and primary tumors and suggested that epigenetic dysregulation may contribute to disease pathogenesis in ES.

Identification of a novel, recurrent SLC44A1-PRKCA fusion in papillary glioneuronal tumor.

Mixed neuronal-glial tumors are rare and challenging to subclassify. One recently recognized variant, papillary glioneuronal tumor (PGNT), is characterized by prominent pseudopapillary structures and glioneuronal elements. We identified a novel translocation, t(9;17)(q31;q24), as the sole karyotypic anomaly in two PGNTs. A fluorescence in situ hybridization (FISH)-based positional cloning strategy revealed SLC44A1, a member of the choline transporter-like protein family, and PRKCA, a protein kinase C family member of serine/threonine-specific protein kinases, as the 9q31 and 17q24 breakpoint candidate genes, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using a forward primer from SLC44A1 exon 5 and a reverse primer from PRKCA exon 10 confirmed the presence of a SLC44A1-PRKCA fusion product in both tumors. Sequencing of each chimeric transcript uncovered an identical fusion cDNA junction occurring between SLC44A1 exon 15 and PRKCA exon 9. A dual-color breakpoint-spanning probe set custom-designed for interphase cell recognition of the translocation event identified the fusion in a third PGNT. These results suggest that the t(9;17)(q31;q24) with the resultant novel fusion oncogene SLC44A1-PRKCA is the defining molecular feature of PGNT that may be responsible for its pathogenesis. The FISH and RT-PCR assays developed in this study can serve as valuable diagnostic adjuncts for this rare disease entity.

Gene-expression signatures differ between different clinical forms of familial hemophagocytic lymphohistiocytosis.

We performed gene-expression profiling of PBMCs obtained from patients with familial hemophagocytic lymphohistiocytosis (FHL) to screen for biologic correlates with the genetic and/or clinical forms of this disease. Unsupervised hierarchical clustering of 167 differentially expressed probe sets, representing 143 genes, identified 3 groups of patients corresponding to the genetic forms and clinical presentations of the disease. Two clusters of up- and down-regulated genes separated patients with perforin-deficient FHL from those with unidentified genetic cause(s) of the disease. The clusterscomprised genes involved in defense/immune responses, apoptosis, zinc homeostasis, and systemic inflammation. Unsupervised hierarchical clustering partitioned patients with unknown genetic cause(s) of FHL into 2 well-distinguished subgroups. Patterns of up- and down-regulated genes separated patients with "late-onset" and "relapsing" forms of FHL from patients with an "early onset and rapidly evolving" form of the disease. A cluster was identified in patients with "late onset and relapsing" form of FHL related to B- and T-cell differentiation/survival, T-cell activation, and vesicular transport. The resulting data suggest that unique gene-expression signatures can distinguish between genetic and clinical subtypes of FHL. These differentially expressed genes may represent biomarkers that can be used as predictors of disease progression.

Somato-dendritic morphology and dendritic signal transfer properties differentiate between fore- and hindlimb innervating motoneurons in the frog Rana esculenta.

BACKGROUND: The location specific motor pattern generation properties of the spinal cord along its rostro-caudal axis have been demonstrated. However, it is still unclear that these differences are due to the different spinal interneuronal networks underlying locomotions or there are also segmental differences in motoneurons innervating different limbs. Frogs use their fore- and hindlimbs differently during jumping and swimming. Therefore we hypothesized that limb innervating motoneurons, located in the cervical and lumbar spinal cord, are different in their morphology and dendritic signal transfer properties. The test of this hypothesis what we report here. RESULTS: Discriminant analysis classified segmental origin of the intracellularly labeled and three-dimensionally reconstructed motoneurons 100% correctly based on twelve morphological variables. Somata of lumbar motoneurons were rounder; the dendrites had bigger total length, more branches with higher branching orders and different spatial distributions of branch points. The ventro-medial extent of cervical dendrites was bigger than in lumbar motoneurons. Computational models of the motoneurons showed that dendritic signal transfer properties were also different in the two groups of motoneurons. Whether log attenuations were higher or lower in cervical than in lumbar motoneurons depended on the proximity of dendritic input to the soma. To investigate dendritic voltage and current transfer properties imposed by dendritic architecture rather than by neuronal size we used standardized distributions of transfer variables. We introduced a novel combination of cluster analysis and homogeneity indexes to quantify segmental segregation tendencies of motoneurons based on their dendritic transfer properties. A segregation tendency of cervical and lumbar motoneurons was detected by the rates of steady-state and transient voltage-amplitude transfers from dendrites to soma at all levels of synaptic background activities, modeled by varying the specific dendritic membrane resistance. On the other hand no segregation was observed by the steady-state current transfer except under high background activity. CONCLUSIONS: We found size-dependent and size-independent differences in morphology and electrical structure of the limb moving motoneurons based on their spinal segmental location in frogs. Location specificity of locomotor networks is therefore partly due to segmental differences in motoneurons driving fore-, and hindlimbs.

Assessment of minimal residual disease in ewing sarcoma.

Advances in molecular pathology now allow for identification of rare tumor cells in cancer patients. Identification of this minimal residual disease is particularly relevant for Ewing sarcoma, given the potential for recurrence even after complete remission is achieved. Using RT-PCR to detect specific tumor-associated fusion transcripts, otherwise occult tumor cells are found in blood or bone marrow in 20-30% of Ewing sarcoma patients, and their presence is associated with inferior outcomes. Although RT-PCR has excellent sensitivity and specificity for identifying tumor cells, technical challenges may limit its widespread applicability. The use of flow cytometry to identify tumor-specific antigens is a recently described method that may circumvent these difficulties. In this manuscript, we compare the advantages and drawbacks of these approaches, present data on a third method using fluorescent in situ hybridization, and discuss issues affecting the further development of these strategies.