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Tert-expressing cells have been identified within the acinar cell compartment of the pancreas in mice. Lineage tracing experiments indicate that these cells can maintain the exocrine compartment during homeostasis and repopulate the tissue during regeneration. Additional expression of mutant Kras in Tert-expressing acinar cells accelerates acinar clone formation and causes transdifferentiation to pre-invasive pancreatic intraepithelial neoplasms after injury. Here, using a genetic approach, we generated mice where tamoxifen injection leads to simultaneous deletion of TERT in Tert-expressing cells and activation of a reporter allele. We performed lineage tracing experiments to investigate the role of TERT in those cells. We studied the effect of acute deletion of TERT during homeostasis, regeneration and tumorigenesis. Acute deletion of TERT significantly inhibited the clone forming capability of Tert-expressing cells during homeostasis in pancreas after one year. To explore the requirement for TERT in an injury setting, we treated mice with cerulein to induce pancreatitis. Somatic inactivagion of TERT in Tert-expressing acinar stem cells impaired clone formation in mice treated with cerulein. To understand if loss of TERT affected transformation, we deleted TERT in acinar stem cells while specifically expressing activated Kras in these cells. Acute deletion of TERT in the presence of a mutant KrasG12D allele decreased the formation of metaplastic areas and PanIN lesions post-injury. These data suggest that a functional allele of Tert is necessary for the clone forming ability of Tert-expressing cells during homeostasis and regeneration as well as acinar to ductal metaplasia and PanIN formation in tumorigenesis. Citation Format: Patrick Neuhöfer, Stewart J. Kim, Gregory W. Charville, Steven E. Artandi. Somatic deletion of Tert inhibits clonal expansion of pancreatic acinar cell stem cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A076.

Soft tissue neoplasms encompass a broad spectrum of clinicopathologic manifestations. In a subset of soft tissue tumors, spanning a wide range of clinical behavior from indolent to highly aggressive, recurrent genetic translocations yield oncogenic fusion proteins that drive neoplastic growth. Beyond functioning as primary mechanisms of tumorigenesis, recurrent translocations represent key diagnostic features insofar as the presence of a particular oncogenic gene fusion generally points to specific tumor entities. In addition to more direct methods for identifying recurrent translocations, such as conventional cytogenetics or fluorescence in situ hybridization, immunohistochemistry for a component of the fusion oncoprotein increasingly is being used as a surrogate marker, exploiting the tendency of these fusion components to be distinctively overexpressed by translocation-bearing tumor cells. Diagnostic immunohistochemistry can also be used to identify the characteristic gene expression changes that occur downstream of oncogenic fusions. Here, we review the use of immunohistochemistry to detect surrogate markers of recurrent translocations in soft tissue tumors, focusing on the practical applications and limitations of this diagnostic approach.

Tenosynovial giant cell tumors (TGCTs) are benign neoplasms that arise from the synovium of tendon sheaths, bursae, and joints. We report a rare presentation of TGCT involving the suboccipital spine.

Alveolar rhabdomyosarcoma (RMS) is associated with an underlying pathogenic translocation involving either PAX3 or PAX7 and FOXO1. The presence or absence of this fusion defines the biology and clinical behavior of this subtype of RMS and its identification in tumors is relevant to prognostication and treatment planning. To further explore the unique characteristics of fusion-driven RMS, we leveraged a published gene expression data set to perform an unbiased comparison of 33 fusion-positive and 25 fusion-negative RMS cases. Our analyses revealed 1790 expressed loci with more than two-fold differential expression at a threshold of P .05. Genes with increased expression in fusion-positive relative to fusion-negative RMS were significantly enriched for those involved in "nervous system development," "neuron differentiation," and "neurogenesis," highlighting a neurodevelopmental gene expression signature driven by the alveolar RMS-associated fusion protein. We show that neurodevelopmental genes are enriched near PAX3-FOXO1 fusion protein binding sites, suggesting a genome-wide fusion protein-mediated activation of cis regulatory elements. Among the genes with differential expression in fusion-positive versus fusion-negative RMS, we identified expression of the transcriptional regulator of motor neuron and oligodendrocyte development, OLIG2, as a marker of the fusion protein-dependent neurodevelopmental signature. Immunohistochemical analysis of a cohort of 73 RMS specimens revealed OLIG2 expression in 96.4% of fusion-positive RMS (N = 27/28), but only in 6.7% of fusion-negative RMS (N = 3/45; P .001). The proportion of OLIG2-expressing cells in fusion-negative cases did not exceed 5%, while 92.9% of fusion-positive cases showed expression in at least 5% of cells. Our findings identify OLIG2 expression as a unique manifestation of a neurodevelopmental gene expression signature driven by the oncogenic fusion protein characteristic of alveolar RMS, which may aid in the diagnostic and prognostic distinction of fusion-positive cases.

Soft tissue sarcomas encompass a broad spectrum of histologically, clinically, and molecularly diverse neoplasms that present unique diagnostic and therapeutic challenges. Accurate classification is essential both for appropriate risk stratification and for guiding clinical management. Once classified almost exclusively based on the morphologic appearance of the tumor by light microscopy, many soft tissue sarcomas are now known to manifest recurrent patterns of genetic alterations. In addition to enabling molecular confirmation of histologic diagnoses, discovery of these recurrent genetic alterations has helped to refine existing morphologic definitions of sarcoma subtypes and even prompted the discovery of new subtypes. As therapy for sarcoma has become increasingly tailored to a specific entity, the integration of molecular data has assumed added importance in diagnostic decision making. In this article, we summarize principles of the histologic evaluation of soft tissue sarcomas, discuss specific diagnostic features of several of the most common sarcoma subtypes, and describe our vision for a future of soft tissue sarcoma diagnosis that merges morphologic, genetic, and epigenetic features to arrive at diagnoses that are aligned with tumor-specific, biologically targeted treatment approaches.

Undifferentiated malignancies (UMs) encompass a diverse set of aggressive tumors that pose not only a diagnostic challenge but also a challenge for clinical management. Most tumors in this category are currently treated empirically with nonspecific chemotherapeutic agents that yield extremely poor clinical response. Given that UMs are inherently genetically unstable neoplasms with the potential for immune dysregulation and increased neoantigen production, they are likely to be particularly amenable to immune checkpoint inhibitors, which target programmed cell death protein 1 (PD-1) or its ligands, PD-L1 and PD-L2, to promote T-cell antitumor activity. Aberrant expression of PD-L1 and, more recently, chromosomal 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) alterations can be used as biomarkers to predict responsiveness to checkpoint inhibitors. Here we evaluated 93 cases previously diagnosed as an "undifferentiated" malignancy and found that 56% (52/93) of UMs moderately to strongly express PD-L1 by immunohistochemistry (IHC). Concurrent CD274(PD-L1) and PDCD1LG2(PD-L2) fluorescence in situ hybridization (FISH) was performed on 24 of these cases and demonstrates a genetic gain at both loci in 62.5% of UMs. Genetic alterations at the CD274(PD-L1) and PDCD1LG2(PD-L2) loci were found to be completely concordant by FISH. Overall, we found that a significant proportion of UMs express PD-L1 and provide molecular support for using checkpoint inhibitors as a treatment approach for this class of tumors.

Platelet-derived growth factor receptor A (PDGFRA) is a receptor tyrosine kinase that is activated by mutations in 10% of gastrointestinal stromal tumors (GISTs) and 55% to 70% of inflammatory fibroid polyps. PDGFRA-mutant GISTs are usually epithelioid and occur predominantly in the stomach. Succinate dehydrogenase-deficient GISTs also arise in the stomach and are usually epithelioid, as are some KIT-mutant GISTs. Recently, avapritinib was approved to treat PDGFRA D842V-mutant GISTs, which do not respond to conventional targeted therapy. Here, we evaluate the utility of PDGFRA immunohistochemistry (IHC) to predict PDGFRA mutations to direct targeted therapy. PDGFRA IHC was performed at 1:3000 and 1:10,000 dilutions on a tissue microarray containing 153 GISTs (126 KIT-mutant, 17 PDGFRA-mutant, and 10 succinate dehydrogenase-deficient). The "positive" staining threshold was defined as 50% of neoplastic cells staining at moderate intensity. PDGFRA IHC was 75.0% and 80.9% specific for PDGFRA mutations at 1:3000 and 1:10,000 dilutions, respectively, and it was 100% sensitive at both. On the basis of its higher specificity, a 1:10,000 dilution was used to stain whole-tissue sections of GISTs and other gastric tumor types. Combining tissue microarray and whole-tissue data, PDGFRA IHC was 94.4% sensitive and 81.0% specific for PDGFRA-mutant GIST among all 210 GISTs, and it was 84.1% specific among 149 GISTs with an epithelioid component. PDGFRA was positive in a subset of inflammatory fibroid polyps (15/30; 50%), monophasic synovial sarcomas (5/10; 50%), inflammatory myofibroblastic tumors (5/10; 50%), and plexiform fibromyxomas (2/8; 25%). It was negative in poorly differentiated adenocarcinoma (0/20), diffuse large B-cell lymphoma (0/10), glomus tumor (0/10), gastrointestinal neuroectodermal tumor (0/10), leiomyoma (0/10), gastric schwannoma (0/8), and gastroblastoma (0/3). Among GISTs, PDGFRA IHC is highly sensitive and moderately specific for PDGFRA-mutant tumors; it also can be positive in inflammatory fibroid polyp and some other mesenchymal tumor types. PDGFRA positivity could be used to triage epithelioid GISTs for PDGFRA sequencing to determine optimal therapy.

Myxoid liposarcoma (MLPS) is a malignant adipocytic neoplasm with predilection for the extremities. MLPS is genetically defined by a t(12;16) translocation leading to FUS-DDIT3 (95%) or more rarely t(12;22) leading to EWSR1-DDIT3. Low-grade MLPS is characterized by bland spindle cells within a myxoid matrix containing delicate "chicken-wire" vasculature, whereas high-grade ("round cell") MLPS may be indistinguishable from other round cell sarcomas. In many cases, cytogenetic or molecular genetic techniques are applied to confirm the diagnosis. A recent study documented the utility of DDIT3 immunohistochemistry (IHC) in the differential diagnosis of adipocytic and myxoid soft tissue tumors. The purpose of this study was to evaluate DDIT3 IHC as a surrogate for molecular testing in high-grade MLPS. IHC was performed using a mouse monoclonal antibody directed against the N-terminus of DDIT3 on whole tissue sections from 50 high-grade MLPS cases and 319 histologic mimics used as controls (170 on whole tissue sections and 149 on a tissue microarray). Histologic mimics included Ewing sarcoma, CIC-rearranged sarcoma, sarcomas with BCOR genetic alterations, poorly differentiated synovial sarcoma, alveolar and embryonal rhabdomyosarcomas, mesenchymal chondrosarcoma, desmoplastic small round cell tumor, and neuroblastoma. Nuclear staining in >5% of cells was considered positive. By IHC, 48 (96%) high-grade MLPS showed strong diffuse nuclear staining for DDIT3. Of the controls, 2% of cases were positive, with no more than 25% nuclear staining. An additional 19% of control cases displayed less than 5% nuclear staining. Overall, DDIT3 IHC showed 96% sensitivity and 98% specificity for high-grade MLPS; strong, diffuse staining is also 96% sensitive but is 100% specific. IHC using an antibody directed against the N-terminus of DDIT3 is highly sensitive and specific for high-grade MLPS among histologic mimics and could replace molecular genetic testing in many cases, although limited labeling may be seen in a range of other tumor types.

Nodular fasciitis is a benign, self-limited, pseudosarcomatous neoplasm that can mimic malignancy due to its rapid growth, cellularity, and mitotic activity. Involvement of the breast is rare and diagnosis on biopsy can be challenging. In this largest series to date, we examined the clinicopathologic and molecular characteristics of 12 cases of nodular fasciitis involving the breast/axilla. All patients were female, with a median age of 32 years (range 15-61). The lesions were 0.4 to 5.8 cm in size (median 0.8). All cases presented as palpable masses, and two patients had overlying skin retraction. Microscopically, lesions were relatively well-circumscribed nodular masses of bland myofibroblastic spindle cells within a variably myxoid stroma. Infiltrative growth into adipose tissue or breast epithelium was frequent. Mitotic figures were present in all cases, ranging from 1 to 12 per 10 high-power fields (median 3). Immunohistochemically, all cases expressed smooth muscle actin and were negative for pan-cytokeratin, p63, desmin, CD34, and nuclear beta-catenin. Targeted RNA sequencing performed on 11 cases identified USP6 gene fusions in eight; one additional case was positive by break-apart fluorescence in situ hybridization. The common MYH9-USP6 rearrangement was detected in four cases; another case had a rare alternative fusion with CTNNB1. Three cases harbored novel USP6 gene fusions involving NACA, SLFN11, or LDHA. All fusions juxtaposed the promoter region of the 5' partner gene with the full-length coding sequence of USP6. Outcome data were available for eight patients; none developed recurrence or metastasis. Five patients elected for observation without immediate excision, and self-resolution of the lesions was reported in three cases. Albeit uncommon, nodular fasciitis should be considered in the differential diagnosis of breast spindle cell lesions. A broad immunohistochemical panel to exclude histologic mimics, including metaplastic carcinoma, is important. Confirmatory detection of USP6 rearrangements can aid in classification, with potential therapeutic implications.

Myxoid liposarcoma is a malignant adipogenic neoplasm characterized by prominent arborizing capillaries, occasional lipoblasts, and primitive-appearing spindle cells in a myxoid background. A recurrent translocation in myxoid liposarcoma results in an oncoprotein consisting of full-length DDIT3 (CHOP) fused to an N-terminal segment of either FUS (TLS) or, less often, EWSR1. Here, we explore the diagnostic significance of DDIT3 expression in myxoid liposarcoma using a mouse monoclonal antibody recognizing an epitope in the N-terminal region. Studying a total of 300 tumors, we find diffuse, moderate-to-strong nuclear-localized anti-DDIT3 immunoreactivity in all 46 cases of myxoid liposarcoma representing 36 unique tumors, including 6 cases with high-grade (round cell) morphology. DDIT3 immunohistochemistry also highlighted a distinctive vasculocentric growth pattern in 7 myxoid liposarcomas treated with neoadjuvant radiation. In contrast, the vast majority of other examined lipomatous and myxoid neoplasms exhibited no DDIT3 expression; limited, weak immunoreactivity in less than 10% of cells was infrequently observed in dedifferentiated liposarcoma (6/39, 15%), solitary fibrous tumor (3/12, 25%), pleomorphic liposarcoma (1/15, 7%), and high-grade myxofibrosarcoma (2/17, 12%). Although this minimal DDIT3 expression did not correlate with DDIT3 amplification or myxoid liposarcoma-like morphology in dedifferentiated liposarcoma, there was evidence among sarcomas (excluding myxoid liposarcoma) of a relationship between expression and exposure to neoadjuvant radiation or cytotoxic chemotherapy. The constellation of findings indicates that DDIT3 immunohistochemistry may have utility in the evaluation of myxoid and lipomatous neoplasms to support the diagnosis of myxoid liposarcoma.

Dermatofibrosarcoma protuberans (DFSP) is a spindle cell neoplasm of the skin and superficial soft tissue with a tendency for locally aggressive behavior; metastatic potential coincides with fibrosarcomatous transformation. The vast majority of DFSPs harbor the t(17;22) translocation resulting in a COL1A1-PDGFB fusion that drives autocrine growth stimulation via PDGFB overexpression. Here, we examined the utility of PDGFB RNA chromogenic in situ hybridization (CISH) for the diagnosis of DFSP. A total of 337 tumors represented in whole tissue sections and tissue microarrays, including 37 cases of DFSP and 300 histologically similar spindle cell tumors, were subjected to PDGFB RNA CISH using commercially available probes. PDGFB overexpression was observed by light microscopy in 24 of 26 conventional DFSPs (92%) and 11 of 11 fibrosarcomatous DFSPs (100%). One of two DFSPs negative for PDGFB by RNA CISH was found to harbor an uncommon alternative rearrangement involving PDGFD. All examined cases of histologic mimics were negative for PDGFB overexpression; limited PDGFB expression, not reaching an empirical threshold of greater than 5 puncta or one aggregate of chromogen in more than 25% of cells, was observed in 7 of 300 mimics (2.3%), including desmoplastic melanoma, malignant peripheral nerve sheath tumor, angiosarcoma, and pleomorphic dermal sarcoma. Vascular PDGFB expression was seen in several tumor types. We conclude that PDGFB RNA CISH, with careful interpretation and the use of appropriate thresholds, may serve as a surrogate marker of PDGFB rearrangement and a useful ancillary tool for the diagnosis of DFSP.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus–mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

Inflammatory leiomyosarcoma (ILMS), defined as "a malignant neoplasm showing smooth muscle differentiation, a prominent inflammatory infiltrate, and near-haploidization", is a very rare soft tissue tumor with a generally favorable prognosis. The morphologic features of "histiocyte-rich rhabdomyoblastic tumor" (HRRMT) are similar to those of ILMS, although this lesion shows by definition a skeletal muscle phenotype. Recent gene expression profiling and immunohistochemical studies have also suggested that ILMS and HRRMT may be related. We studied the clinicopathologic, immunohistochemical and genetic features of four cases previously classified as ILMS and nine classified as HRRMT. Tumors from both groups tended to occur in the deep soft tissues of the extremities of young to middle-aged males and exhibited indolent behavior. Morphologically, all were well-circumscribed, often encapsulated, and showed a striking histiocyte-rich inflammatory infiltrate admixed with variably pleomorphic tumor cells showing spindled and epithelioid to rhabdoid morphology, eosinophilic cytoplasm, and prominent nucleoli, but few, if any, mitotic figures. Immunohistochemically, the tumor cells expressed desmin, alpha-smooth muscle actin, and the rhabdomyoblastic markers PAX7, MyoD1, and myogenin. H-caldesmon expression was absent in all cases, using the specific h-CD antibody. Karyotypic study (1 HRRMT) and genome-wide copy number analysis (7 HRRMT, OncoScan SNP assay), revealed near-haploidization in four cases, with subsequent genome doubling in one, an identical phenotype to that seen in ILMS. We propose reclassification of ILMS and HRRMT as "inflammatory rhabdomyoblastic tumor", a name which accurately describes the salient morphologic and immunohistochemical features of this distinctive tumor, as well as its intermediate (rarely metastasizing) clinical behavior.

Chondroblastoma is currently classified as a benign neoplasm; however, chondroblastoma and chondroblastoma-like osteosarcoma have morphologic overlap, raising the possibility that some tumors diagnosed as chondroblastoma-like osteosarcoma might actually represent malignant chondroblastoma. The H3F3B K36M point mutation, which has not been reported in osteosarcoma, is identified in 95% of chondroblastomas and is reliably detectable by immunohistochemistry (IHC). We reviewed 11 tumors diagnosed as atypical chondroblastoma, malignant chondroblastoma, or chondroblastoma-like osteosarcoma (median follow-up: 8.8 years; range: 4 months-26.4 years). Seven chondroblastomas with cytologic atypia and permeative growth were designated "malignant chondroblastoma"; six were H3K36M-positive by IHC. Relative to conventional chondroblastoma, malignant chondroblastoma occurred in older individuals (median: 52 years; range: 29-57 years) and arose at unusual sites. Three of four tumors with long-term follow-up recurred, and one patient died of widespread metastases. One was found to have chromosomal copy number alter4ations and a SETD2 mutation in addition to H3F3B K36M. The four remaining tumors were classified as chondroblastoma-like osteosarcoma. Chondroblastoma-like osteosarcoma occurred in younger patients (median: 21 years; range: 19-40 years) than malignant chondroblastoma. In contrast to malignant chondroblastoma, all had regions of malignant cells forming bone. Two of three patients with long-term follow-up developed recurrences, and two died of disease, one with widespread metastases. No mutations in H3F3A/H3F3B were detected by Sanger sequencing. While malignant chondroblastoma and chondroblastoma-like osteosarcoma show significant morphologic overlap, they have distinct clinical presentations and genetic findings. When considering this challenging differential diagnosis, IHC using histone H3 mutation-specific antibodies is a critical diagnostic adjunct.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines., Graphical Abstract

Noninfectious gastrointestinal (GI) vasculopathic disorders are rare and are often overlooked in histopathologic examination or when forming differential diagnoses due to their rarity. However, involvement of the GI tract may lead to serious complications, including ischemia and perforation. Since awareness of the types of vasculopathy that may involve the GI tract is central to arriving at a correct diagnosis, we reviewed our institutional experience with GI tract vasculopathy in order to enhance diagnostic accuracy of these rare lesions. We report the clinical and histologic features of 16 cases (excluding 16 cases of immunoglobulin A vasculitis) diagnosed over a 20-year period. Of the 16 patients, 14 presented with symptoms related to the GI vasculopathy (including 2 presenting with a mass on endoscopic examination). The remaining 2 patients presented with incarcerated hernia and invasive adenocarcinoma. The vasculopathy was not associated with systemic disease and appeared limited to the GI tract in 8 patients. Eight had associated systemic disease, but only 6 had a prior diagnosis. The underlying diagnoses in these 6 patients included systemic lupus erythematosus (1), dermatomyositis (2), rheumatoid arthritis (1), eosinophilic granulomatosis with polyangiitis (1), and Crohn disease (1). One patient with granulomatous polyangiitis and 1 patient with systemic lupus erythematosus initially presented with GI symptoms. The 8 cases of isolated GI tract vasculopathy consisted of enterocolic lymphocytic phlebitis (4), idiopathic myointimal hyperplasia of the sigmoid colon (1), idiopathic myointimal hyperplasia of the ileum (1), granulomatous vasculitis (1), and polyarteritis nodosa-like arteritis (1). Isolated GI tract vasculopathy is rare, but appears to be almost as common as that associated with systemic disease. The chief primary vasculopathies are enterocolic lymphocytic colitis and idiopathic myointimal hyperplasia. Although the latter occurs predominantly in the left colon, rare examples occur in the small bowel and likely represent a complex, more protean disorder.

PAX7 is a paired-box transcription factor that is required for the developmental specification of adult skeletal muscle progenitors in mice. We previously demonstrated PAX7 expression as a marker of skeletal muscle differentiation in rhabdomyosarcoma. Here, using analyses of published whole-genome gene expression microarray data, we identify PAX7 as a gene with significantly increased expression in Ewing sarcoma in comparison to CIC-DUX4 round cell sarcoma. Analysis of PAX7 in a large cohort of 103 Ewing sarcoma cases by immunohistochemistry revealed expression in 99.0% of cases (102/103). PAX7 expression was noted in cases demonstrating three distinct Ewing sarcoma EWSR1 translocations involving FLI1, ERG, and NFATc2. No PAX7 expression was observed in any of 27 cases of CIC-DUX4 sarcoma by immunohistochemistry (0%; 0/27). Exploring the mechanism of PAX7 expression in Ewing sarcoma using curated RNA- and ChIP-sequencing data, we demonstrate that the EWSR1 fusion protein is required for PAX7 expression in Ewing sarcoma and identify a candidate EWSR1-FLI1-bound PAX7 enhancer that coincides with both a consensus GGAA repeat-containing binding site and a peak of regulatory H3K27 acetylation. Taken together, our findings provide mechanistic support for the utility of PAX7 immunohistochemistry in the diagnosis of Ewing sarcoma, while linking this sarcoma of uncertain histogenesis to a key transcriptional regulator of mammalian muscle progenitor cells.

Insulinoma-associated protein 1 (INSM1) is a transcription factor that functions in neuroepithelial tissue development and shows expression in neuroendocrine neoplasms. Given the role of INSM1 in controlling differentiation of the sympatho-adrenal lineage, we hypothesized that INSM1 expression would define a subset of neuroblastic tumors. This study aimed to characterize the immunohistochemical profile of INSM1 in a cohort of peripheral neuroblastic tumors and compare INSM1 expression in these tumors to that seen in other embryonal neoplasms, using both tissue microarrays and whole-slide histologic sections. INSM1 showed nuclear expression in 39/50 (78%) peripheral neuroblastic tumors, including 27/32 (84%) neuroblastomas, 9/9 (100%) ganglioneuroblastomas, and 3/9 (33%) ganglioneuromas. Altogether, 70% of peripheral neuroblastic tumors showed anti-INSM1 immunoreactivity in more than 20% of tumor nuclei. Although no non-neuroblastic tumors in this study exhibited INSM1 expression in more than 20% of nuclei, focal or patchy staining was identified in 7/14 (50%) rhabdomyosarcomas, 7/22 (32%) nephroblastomas, and 4/20 (20%) Ewing sarcomas. The absence of INSM1 expression in peripheral neuroblastic tumors was restricted to undifferentiated and poorly differentiated neuroblastomas, as well as mature ganglioneuromas, mimicking the transient INSM1 expression seen in sympatho-adrenal differentiation during normal development. No significant association between MYCN amplification status and INSM1 expression was observed. We found that all 3 INSM1-negative neuroblastoma patients with available follow-up were alive at a median of 15 years, in comparison to 9 of 13 INSM1-positive neuroblastoma patients living at a median of 5 years. Additional studies are needed to determine whether INSM1 expression is indicative of a clinically significant differentiation state in neuroblastoma.

Serous neoplasms are uncommon, usually cystic tumors that account for less than 1% of all primary pancreatic lesions. They consist predominantly of a monomorphic epithelial cell population with a glycogen-rich, clear cytoplasm, reminiscent of clear cell renal cell carcinoma, with which serous neoplasms share an association with underlying VHL loss-of-function mutations. Serous neoplasms have no metastatic potential. Accurate recognition of this entity, including its various architectural subtypes, is critical to appropriate prognostication and treatment. Immunohistochemical detection of inhibin and calponin expression, along with the absence of both estrogen and progesterone receptors and nuclear β-catenin, can help to distinguish serous neoplasms from mimics. With the advent of minimally invasive and molecularly driven diagnostic techniques, the pathologist's role in the assessment and management of serous neoplasms has become increasingly complex and important. We provide an update on the histologic, immunohistochemical, and molecular features of pancreatic serous neoplasms for the practicing pathologist.

PAX7 has been recently demonstrated to be a highly sensitive marker for Ewing sarcoma, and thus far has only been shown to label a relatively small set of other mesenchymal neoplasms. Because the processing of bone marrow core biopsies can often hinder the performance of immunohistochemical stains, we set out to determine if our laboratory's PAX7 staining protocol effectively detects Ewing sarcoma in Bouin's fixed, decalcified bone marrow core biopsies. We stained ten core biopsies involved by Ewing sarcoma, nine non-involved core biopsies, and 13 core biopsies involved by histologic mimics of Ewing sarcoma. Only the ten biopsies involved by Ewing sarcoma and four biopsies with rhabdomyosarcoma showed strong nuclear PAX7 staining. None of the other tumors demonstrated PAX7 expression. This study demonstrates that the PAX7 staining protocol used in our laboratory is a useful marker for Ewing sarcoma and other PAX7-positive tumors in decalcified bone marrow core biopsies.

Summary The decline of tissue regenerative potential with age correlates with impaired stem cell function. However, limited strategies are available for therapeutic modulation of stem cell function during aging. Using skeletal muscle stem cells (MuSCs) as a model system, we identify cell death by mitotic catastrophe as a cause of impaired stem cell proliferative expansion in aged animals. The mitotic cell death is caused by a deficiency in Notch activators in the microenvironment. We discover that ligand-dependent stimulation of Notch activates p53 in MuSCs via inhibition of Mdm2 expression through Hey transcription factors during normal muscle regeneration and that this pathway is impaired in aged animals. Pharmacologic activation of p53 promotes the expansion of aged MuSCs in vivo. Altogether, these findings illuminate a Notch-p53 signaling axis that plays an important role in MuSC survival during activation and is dysregulated during aging, contributing to the age-related decline in muscle regenerative potential.

Synovial sarcoma (SS) is defined by the hallmark SS18-SSX fusion oncoprotein, which renders BAF complexes aberrant in two manners: gain of SSX to the SS18 subunit and concomitant loss of BAF47 subunit assembly. Here we demonstrate that SS18-SSX globally hijacks BAF complexes on chromatin to activate an SS transcriptional signature that we define using primary tumors and cell lines. Specifically, SS18-SSX retargets BAF complexes from enhancers to broad polycomb domains to oppose PRC2-mediated repression and activate bivalent genes. Upon suppression of SS18-SSX, reassembly of BAF47 restores enhancer activation, but is not required for proliferative arrest. These results establish a global hijacking mechanism for SS18-SSX on chromatin, and define the distinct contributions of two concurrent BAF complex perturbations.

Summary Adult skeletal muscle stem cells, or satellite cells (SCs), regenerate functional muscle following transplantation into injured or diseased tissue. To gain insight into human SC (huSC) biology, we analyzed transcriptome dynamics by RNA sequencing of prospectively isolated quiescent and activated huSCs. This analysis indicated that huSCs differentiate and lose proliferative potential when maintained in high-mitogen conditions ex vivo. Further analysis of gene expression revealed that p38 MAPK acts in a transcriptional network underlying huSC self-renewal. Activation of p38 signaling correlated with huSC differentiation, while inhibition of p38 reversibly prevented differentiation, enabling expansion of huSCs. When transplanted, expanded huSCs differentiated to generate chimeric muscle and engrafted as SCs in the sublaminar niche with a greater frequency than freshly isolated cells or cells cultured without p38 inhibition. These studies indicate characteristics of the huSC transcriptome that promote expansion ex vivo to allow enhanced functional engraftment of a defined population of self-renewing huSCs., Graphical Abstract, Highlights • Prospective isolation of highly pure huSCs from diverse muscles • RNA sequencing resource for studying the huSC transcriptome • Core transcription factor regulatory network of huSC differentiation • Expanded huSCs that are genetically manipulable and self-renew in vivo, In this article, Rando and colleagues purify and study human skeletal muscle stem cells using RNA sequencing and cell transplantation. They show that p38 MAPK signaling is important for human muscle stem cell differentiation and that pharmacologic inhibition of p38 enables expansion of muscle stem cells capable of self-renewing after transplantation.

Gastrointestinal stromal tumor (GIST), the most common mesenchymal neoplasm of the gastrointestinal tract, exhibits diverse histologic and clinical manifestations. With its putative origin in the gastrointestinal pacemaker cell of Cajal, GIST can arise in association with any portion of the tubular gastrointestinal tract. Morphologically, GISTs are classified as spindled or epithelioid, though each of these subtypes encompasses a broad spectrum of microscopic appearances, many of which mimic other histologic entities. Despite this morphologic ambiguity, the diagnosis of GIST is aided in many cases by immunohistochemical detection of KIT (CD117) or DOG1 expression. The natural history of GIST ranges from that of a tumor cured by surgical resection to that of a locally advanced or even widely metastatic, and ultimately fatal, disease. This clinicopathologic heterogeneity is paralleled by an underlying molecular diversity: the majority of GISTs are associated with spontaneous activating mutations in KIT, PDGFRA, or BRAF, while additional subsets are driven by genetic lesions-often inherited-of NF1 or components of the succinate dehydrogenase enzymatic complex. Specific gene mutations correlate with particular anatomic or morphologic characteristics and, in turn, with distinct clinical behaviors. Therefore, prognostication and treatment are increasingly dictated not only by morphologic clues, but also by accompanying molecular genetic features. In this review, we provide a comprehensive description of the heterogenous molecular underpinnings of GIST, including implications for the practicing pathologist with regard to morphologic identification, immunohistochemical diagnosis, and clinical management.

If a eukaryotic cell is to reproduce, it must duplicate its genetic information in the form of DNA, and faithfully segregate that information during a complex process of cell division. During this division process, the resulting cells inherit one, and only one, copy of each chromosome. Over thirty years ago, it was predicted that the segregation of sister chromosomes could occur non-randomly, such that a daughter cell would preferentially inherit one of the two sister chromosomes according to some characteristic of that chromosome’s template DNA strand. Although this prediction has been confirmed in studies of various cell-types, we know little of both the mechanism by which the asymmetric inheritance occurs and the significance it has to cells. In this essay, we propose a new model of non-random chromosome segregation – the mortal strand hypothesis – and discuss tests of the model that will provide insight into the molecular choreography of this intriguing phenomenon.

Summary The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.

Rhabdomyosarcoma, the most common soft tissue malignancy of childhood, is a morphologically variable tumor defined by its phenotype of skeletal muscle differentiation. The diagnosis of rhabdomyosarcoma often relies in part on the identification of myogenic gene expression using immunohistochemical or molecular techniques. However, these techniques show imperfect sensitivity and specificity, particularly in scant tissue biopsies. Here, we expand the toolkit for rhabdomyosarcoma diagnosis by studying the expression of PAX7, a transcriptional regulator of mammalian muscle progenitor cells implicated in the pathogenesis of rhabdomyosarcoma. Immunohistochemical analysis of tissue microarrays using a monoclonal anti-PAX7 antibody was used to characterize PAX7 expression in 25 non-neoplastic tissues, 109 rhabdomyosarcomas, and 697 small round blue cell or other soft tissue tumors. Among non-neoplastic tissues, PAX7 was specifically expressed in adult muscle progenitor cells (satellite cells). In embryonal rhabdomyosarcoma, PAX7 expression was positive in 52 of 63 cases (83%), negative in 9 of 63 cases (14%), and focal in 2 of 63 cases (3%). PAX7-positive embryonal rhabdomyosarcoma cases included several showing focal or negative myogenin expression. PAX7 expression in alveolar rhabdomyosarcoma was positive in 6 of 31 cases (19%), negative in 14 of 31 cases (45%), and focal in 11 of 31 cases (36%). In addition, PAX7 was expressed in 5 of 7 pleomorphic rhabdomyosarcomas (71%) and 6 of 8 spindle cell rhabdomyosarcomas (75%). Among histologic mimics, only Ewing sarcoma showed PAX7 expression (7/7 cases, 100%). In contrast, expression of PAX7 was not seen in the large majority (688/690, 99.7%) of examined cases of other soft tissue tumors, small round blue cell neoplasms, and leukemias/lymphomas. In summary, immunohistochemical analysis of PAX7 expression may be a useful diagnostic tool in the assessment of skeletal muscle differentiation in human tumors.

Adult stem cells maintain the mature tissues of metazoans. They do so by reproducing in such a way that their progeny either differentiate, and thus contribute functionally to a tissue, or remain uncommitted and replenish the stem cell pool. Because ageing manifests as a general decline in tissue function, diminished stem cell-mediated tissue maintenance may contribute to age-related pathologies. Accordingly, the mechanisms by which stem cell regenerative potential is sustained, and the extent to which these mechanisms fail with age, are fundamental determinants of tissue ageing. Here, we explore the mechanisms of asymmetric division that account for the sustained fitness of adult stem cells and the tissues that comprise them. In particular, we summarize the theory and experimental evidence underlying non-random chromosome segregation—a mitotic asymmetry arising from the unequal partitioning of chromosomes according to the age of their template DNA strands. Additionally, we consider the possible consequences of non-random chromosome segregation, especially as they relate to both replicative and chronological ageing in stem cells. While biased segregation of chromosomes may sustain stem cell replicative potential by compartmentalizing the errors derived from DNA synthesis, it might also contribute to the accrual of replication-independent DNA damage in stem cells and thus hasten chronological ageing.

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Tissues throughout the body share a common systemic environment comprising the multitude of circulating factors transported within the vasculature. These circulating factors, including hormones and nutrients, represent many key determinants of cell growth and function. As animals age, the balance of factors in the systemic circulation shifts and thus alters the cellular environment. The age-dependent changes in the systemic circulation exposes tissues, including skin, to a distinct combination of factors that is reflected by alterations in cell and tissue phenotypes with age. This chapter discusses our current understanding of the changes to the systemic milieu that occur as mammals grow old, the influence of these changes on skin health and disease in aged individuals, and the efforts to therapeutically modulate circulating factors to prevent or reverse aging phenotypes.

A xerogel-based optical pH sensor capable of releasing low levels of nitric oxide (NO) and measuring changes in solution pH is reported. Through simple dip-coating procedures, aminoalkoxysilane-based xerogel films modified with N-diazeniumdiolate NO donor precursors and the fluorescent pH indicator seminaphthorhodamine-1 carboxylate (SNARF-1) were sequentially deposited onto optical fibers. The resulting sensors were characterized by fast and linear response to pH throughout the physiological range (pH 7.0-7.8). Real-time chemiluminescence measurements confirmed that the presence of the overlying SNARF-1-containing TMOS layer did not have an inhibitory effect on N-diazeniumdiolate formation or NO release, and the NO-releasing coatings were capable of maintaining NO fluxes0.4 pmol/cm(2) s up to 16 h. In vitro blood compatibility studies using porcine platelets confirmed the expected thromboresistivity of the NO-releasing xerogel coatings.

Our goal is to develop an AAV vector that can transduce human skeletal muscle after intramuscular administration at levels sufficient to express therapeutic levels of antibodies with broad-spectrum protection against HIV. AAV has begun being used in passive vaccines for HIV and influenza. Yet for many indications, greater human skeletal muscle transduction is needed than has been achieved with existing AAV serotypes. To bioengineer novel capsids with unparalleled human skeletal muscle transduction, we utilized primary human skeletal muscle cells from surgical resections to screen libraries of replication-competent shuffled AAV capsids. Two screens were performed in pools of primary human skeletal muscle stem cells or myotubes from six patients. Six rounds of replicating selection were carried out and the five most highly selected variants from each screen were vectorized and validated against existing serotypes with muscle tropism (AAV1, 6 and 8). In primary human muscle stem cells, variants NP22, NP66 and NP94 had significantly increased transduction that ranged from a 65 to 284-fold improvement over AAV1, 27 to 118-fold over AAV6, and 10 to 45-fold over AAV8. In primary human myotubes, NP22, NP66 and NP94 again showed significantly increased transduction that ranged from a 13 to 464-fold improvement over AAV1, 25 to 871-fold over AAV8, and NP94 showed a 15-fold improvement over AAV6. To assess human skeletal muscle transduction capabilities in vivo, we injected xenografted humanized muscle NSG mice with shuffled or control capsids expressing luciferase by intramuscular injection (1E9/leg) and assessed transduction weekly by live imaging in time course studies over two months. AAV6 was the first to uncoat but NP66 produced the highest sustained transduction levels. To control for species-specific transduction, we performed the same time course experiment in strain- and gender-matched non-transplanted mice. Strikingly, AAV6 outperformed shuffled variants when no human muscle fibers were present, highlighting the importance of performing preclinical studies in human cells and xenograft systems whenever possible to prevent misleading results which are mouse-specific. To further demonstrate the superiority of our shuffled variants and more accurately predict eventual muscle transduction in humans, we transduced human skeletal muscle explants from surgical resections ex vivo. Four adult patients (two male and two female) had healthy skeletal muscle tissue removed for ex vivo transduction analyses with luciferase. In all 4 patients, NP22 and NP66 (and NP94 in 3/4 patients) had significantly increased transduction by live fiber luciferase imaging as well as luciferase assays on lysed fibers that ranged from a 4 to 116-fold improvement over all control serotypes. To support preclinical vaccine testing in non-human primates, we assessed transduction in rhesus macaque skeletal muscle explants ex vivo. Here again we showed that NP22 and NP66 showed a 30 to 57-fold improvement in transduction over control serotypes. Taken together, our results demonstrate that capsid variants NP22 and NP66 (and in some cases NP94), have highly significant increased human skeletal muscle fiber transduction when assessed in vitro, in vivo and ex vivo. These capsids represent powerful tools to express therapeutic quantities of human monoclonal antibodies for use in passive vaccines, or transgenes for various muscle disorders in gene therapy, specifically in humans.

A mouse study reveals that the stem cell quiescent state is composed of two distinct phases, G0 and GAlert; stem cells reversibly transition between these two phases in response to systemic environmental stimuli acting through the mTORC1 pathway. This paper demonstrates that the in vivo stem-cell quiescent state is composed of distinct phases, much like the phases of the cell cycle. Thomas Rando and colleagues studied stem-cell quiescence in muscle stem cells in vivo and identified a quiescent phase that they call GAlert, which is distinct from the familiar G0 state and is stable, reversible and regulated by environmental stimuli through mTORC1. The authors propose that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism by which stem cells can rapidly respond to injury and stress without requiring cell cycle entry or a cell fate commitment. A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state1. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function2. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an ‘alert’ phase we term GAlert. Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into GAlert and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an ‘alerting’ mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.

Among the key properties that distinguish adult mammalian stem cells from their more differentiated progeny is the ability of stem cells to remain in a quiescent state for prolonged periods of time. However, the molecular pathways for the maintenance of stem-cell quiescence remain elusive. Here we use adult mouse muscle stem cells (satellite cells) as a model system and show that the microRNA (miRNA) pathway is essential for the maintenance of the quiescent state. Satellite cells that lack a functional miRNA pathway spontaneously exit quiescence and enter the cell cycle. We identified quiescence-specific miRNAs in the satellite-cell lineage by microarray analysis. Among these, miRNA-489 (miR-489) is highly expressed in quiescent satellite cells and is quickly downregulated during satellite-cell activation. Further analysis revealed that miR-489 functions as a regulator of satellite-cell quiescence, as it post-transcriptionally suppresses the oncogene Dek, the protein product of which localizes to the more differentiated daughter cell during asymmetric division of satellite cells and promotes the transient proliferative expansion of myogenic progenitors. Our results provide evidence of the miRNA pathway in general, and of a specific miRNA, miR-489, in actively maintaining the quiescent state of an adult stem-cell population.

The ability of nitric oxide (NO)-releasing xerogels to reduce fibrinogen-mediated adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli is described. A negative correlation was observed between NO surface flux and bacterial adhesion for each species tested. For S. aureus and E. coli, reduced adhesion correlated directly with NO flux from 0 to 30 pmol cm(-2)s(-1). A similar dependence for S. epidermidis was evident from 18 to 30 pmol cm(-2)s(-1). At a NO flux of 30 pmol cm(-2)s(-1), surface coverage of S. aureus, S. epidermidis, and E. coli was reduced by 96, 48, and 88%, respectively, compared to non-NO-releasing controls. Polymeric NO release was thus demonstrated to be an effective approach for significantly reducing fibrinogen-mediated adhesion of both gram-positive and gram-negative bacteria in vitro, thereby illustrating the advantage of active NO release as a strategy for inhibiting bacterial adhesion in the presence of pre-adsorbed protein.