A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

Restricted Versus Liberal Versus Goal-Directed Fluid Therapy for Non-vascular Abdominal Surgery: A Network Meta-Analysis and Systematic Review.

Optimal perioperative fluid management is crucial, with over- or under-replacement associated with complications. There are many strategies for fluid therapy, including liberal fluid therapy (LFT), restrictive fluid therapy (RFT) and goal-directed fluid therapy (GDT), without a clear consensus as to which is better. We aimed to find out which is the more effective fluid therapy option in adult surgical patients undergoing non-vascular abdominal surgery in the perioperative period. This study is a systematic review and network meta-analysis (NMA) with node-splitting analysis of inconsistency, sensitivity analysis and meta-regression. We conducted a literature search of Pubmed, Cochrane Library, EMBASE, Google Scholar and Web of Science. Only studies comparing restrictive, liberal and goal-directed fluid therapy during the perioperative phase in major non-cardiac surgery in adult patients will be included. Trials on paediatric patients, obstetric patients and cardiac surgery were excluded. Trials that focused on goal-directed therapy monitoring with pulmonary artery catheters and venous oxygen saturation (SvO2), as well as those examining purely biochemical and laboratory end points, were excluded. A total of 102 randomised controlled trials (RCTs) and 78 studies (12,100 patients) were included. NMA concluded that goal-directed fluid therapy utilising FloTrac was the most effective intervention in reducing the length of stay (LOS) (surface under cumulative ranking curve (SUCRA) = 91%, odds ratio (OR) = -2.4, 95% credible intervals (CrI) = -3.9 to -0.85) and wound complications (SUCRA = 86%, OR = 0.41, 95% CrI = 0.24 to 0.69). Goal-directed fluid therapy utilising pulse pressure variation was the most effective in reducing the complication rate (SUCRA = 80%, OR = 0.25, 95% CrI = 0.047 to 1.2), renal complications (SUCRA = 93%, OR = 0.23, 95% CrI = 0.045 to 1.0), respiratory complications (SUCRA = 74%, OR = 0.42, 95% CrI = 0.053 to 3.6) and cardiac complications (SUCRA = 97%, OR = 0.067, 95% CrI = 0.0058 to 0.57). Liberal fluid therapy was the most effective in reducing the mortality rate (SUCRA = 81%, OR = 0.40, 95% CrI = 0.12 to 1.5). Goal-directed therapy utilising oesophageal Doppler was the most effective in reducing anastomotic leak (SUCRA = 79%, OR = 0.45, 95% CrI = 0.12 to 1.5). There was no publication bias, but moderate to substantial heterogeneity was found in all networks. In preventing different complications, except mortality, goal-directed fluid therapy was consistently more highly ranked and effective than standard (SFT), liberal or restricted fluid therapy. The evidence grade was low quality to very low quality for all the results, except those for wound complications and anastomotic leak.

MASP2 inhibition by narsoplimab suppresses endotheliopathies characteristic of transplant-associated thrombotic microangiopathy: in vitro and ex vivo evidence.

Transplant-associated thrombotic microangiopathy (TA-TMA) is an endotheliopathy complicating up to 30% of allogeneic hematopoietic stem cell transplants (alloHSCT). Positive feedback loops among complement, pro-inflammatory, pro-apoptotic, and coagulation cascade likely assume dominant roles at different disease stages. We hypothesized that mannose-binding lectin-associated serine protease 2 (MASP2), principal activator of the lectin complement system, is involved in the microvascular endothelial cell (MVEC) injury characteristic of TA-TMA through pathways that are susceptible to suppression by anti-MASP2 monoclonal antibody narsoplimab. Pre-treatment plasmas from 8 of 9 TA-TMA patients achieving a complete TMA response in a narsoplimab clinical trial activated caspase 8, the initial step in apoptotic injury, in human MVEC. This was reduced to control levels following narsoplimab treatment in 7 of the 8 subjects. Plasmas from 8 individuals in an observational TA-TMA study, but not 8 alloHSCT subjects without TMA, similarly activated caspase 8, which was blocked in vitro by narsoplimab. mRNA sequencing of MVEC exposed to TA-TMA or control plasmas with and without narsoplimab suggested potential mechanisms of action. The top 40 narsoplimab-affected transcripts included upregulation of SerpinB2, which blocks apoptosis by inactivating procaspase 3; CHAC1, which inhibits apoptosis in association with mitigation of oxidative stress responses; and pro-angiogenesis proteins TM4SF18, ASPM, and ESM1. Narsoplimab also suppressed transcripts encoding pro-apoptotic and pro-inflammatory proteins ZNF521, IL1R1, Fibulin-5, aggrecan, SLC14A1, and LOX1, and TMEM204, which disrupts vascular integrity. Our data suggest benefits to narsoplimab use in high-risk TA-TMA and provide a potential mechanistic basis for the clinical efficacy of narsoplimab in this disorder.

OX40L-expressing recombinant modified vaccinia virus Ankara induces potent antitumor immunity via reprogramming Tregs.

Effective depletion of immune suppressive regulatory T cells (Tregs) in the tumor microenvironment without triggering systemic autoimmunity is an important strategy for cancer immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus with a long history of human use. Here, we report rational engineering of an immune-activating recombinant MVA (rMVA, MVA∆E5R-Flt3L-OX40L) with deletion of the vaccinia E5R gene (encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase, cGAS) and expression of two membrane-anchored transgenes, Flt3L and OX40L. Intratumoral (IT) delivery of rMVA (MVA∆E5R-Flt3L-OX40L) generates potent antitumor immunity, dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and type I IFN signaling. Remarkably, IT rMVA (MVA∆E5R-Flt3L-OX40L) depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Single-cell RNA-seq analyses of tumors treated with rMVA showed the depletion of OX40hiCCR8hi Tregs and expansion of IFN-responsive Tregs. Taken together, our study provides a proof-of-concept for depleting and reprogramming intratumoral Tregs via an immune-activating rMVA.

Defibrotide mitigates endothelial cell injury induced by plasmas from patients with COVID-19 and related vasculopathies.

BACKGROUND AND OBJECTIVES: COVID-19 progression is characterized by systemic small vessel arterial and venous thrombosis. Microvascular endothelial cell (MVEC) activation and injury, platelet activation, and histopathologic features characteristic of acute COVID-19 also describe certain thrombotic microangiopathies, including atypical hemolytic-uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hematopoietic stem cell transplant (HSCT)-associated veno-occlusive disease (VOD). We explored the effect of clinically relevant doses of defibrotide, approved for HSCT-associated VOD, on MVEC activation/injury. METHODS: Human dermal MVEC were exposed to plasmas from patients with acute TMAs or acute COVID-19 in the presence and absence of defibrotide (5µg/ml) and caspase 8, a marker of EC activation and apoptosis, was assessed. RNAseq was used to explore potential mechanisms of defibrotide activity. RESULTS: Defibrotide suppressed TMA plasma-induced caspase 8 activation in MVEC (mean 60.2 % inhibition for COVID-19; p = 0.0008). RNAseq identified six major cellular pathways associated with defibrotide's alteration of COVID-19-associated MVEC changes: TNF-α signaling; IL-17 signaling; extracellular matrix (ECM)-EC receptor and platelet receptor interactions; ECM formation; endothelin activity; and fibrosis. Communications across these pathways were revealed by STRING analyses. Forty transcripts showing the greatest changes induced by defibrotide in COVID-19 plasma/MVEC cultures included: claudin 14 and F11R (JAM), important in maintaining EC tight junctions; SOCS3 and TNFRSF18, involved in suppression of inflammation; RAMP3 and transgelin, which promote angiogenesis; and RGS5, which regulates caspase activation and apoptosis. CONCLUSION: Our data, in the context of a recent clinical trial in severe COVID-19, suggest benefits to further exploration of defibrotide and these pathways in COVID-19 and related endotheliopathies.

Discovery of a Vertebral Skeletal Stem Cell Driving Spinal Metastases.

Vertebral bone is subject to a distinct set of disease processes from those of long bones, notably including a much higher rate of solid tumor metastases that cannot be explained by passive blood flow distribution alone. The basis for this distinct biology of vertebral bone has remained elusive. Here we identify a vertebral skeletal stem cell (vSSC), co-expressing the transcription factors ZIC1 and PAX1 together with additional cell surface markers, whose expression profile and function are markedly distinct from those of long bone skeletal stem cells (lbSSCs). vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. Lineage tracing of vSSCs confirms that they make a persistent contribution to multiple mature cell lineages in the native vertebrae. vSSCs are physiologic mediators of spine mineralization, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed clinically in breast cancer. Specifically, when an organoid system is used to place both vSSCs and lbSSCs in an identical anatomic context, vSSC-lineage cells are more efficient than lbSSC-lineage cells at recruiting metastases, a phenotype that is due in part to increased secretion of the novel metastatic trophic factor MFGE8. Similarly, genetically targeting loss-of-function to the vSSC lineage results in reduced metastasis rates in the native vertebral environment. Taken together, vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of metastatic seeding of the vertebrae.

Disseminated Mucocutaneous Histoplasmosis Diagnosed in the United Kingdom, Presumably as a Result of Recrudescence Decades After Primary Infection Following Immunosuppressive Treatment of Its Mimic, Sarcoidosis: A Multidisciplinary Cautionary Tale.

ABSTRACT: Histoplasmosis is a dimorphic fungal infection, which is rare outside endemic pockets in North, Central, and South America, Asia, and Africa. Herein, we describe a woman in her 80s living in the Scottish Borders region of the United Kingdom with a recent diagnosis of granulomatous rosacea, who on receiving escalating immunosuppression for suspected sarcoidosis, and long-standing rheumatoid arthritis developed a striking eruption involving her eyelids along with painful ulceration of the oral and nasal mucosa. Histopathologic examination of the skin and mucosal lesions demonstrated granulomatous inflammation with numerous yeast forms of fungal organisms with morphological characteristics of Histoplasma species. This was confirmed to be H. capsulatum on fungal culture and direct panfungal polymerase chain reaction assay. Although the patient had not left the United Kingdom for more than 20 years, she gave a travel history involving multiple trips to countries where histoplasmosis is known to occur, before that. This case exemplifies the challenges involved in making a diagnosis of histoplasmosis in nonendemic regions for both clinicians and pathologists alike. In this particular patient, the diagnostic difficulties were compounded by the clinicopathological overlap with other cutaneous and systemic granulomatous disorders like granulomatous rosacea and suspected sarcoidosis and also the exceptionally long latency period between the purported historical primary infection and recent recrudescence. We highlight this unusual case to increase an awareness of histoplasmosis, which is very rare in nonendemic regions like the United Kingdom and involves cases acquired during residence in or travel to endemic areas, to ensure its prompt recognition and treatment.

Low-dose carbon monoxide suppresses metastatic progression of disseminated cancer cells.

Low-dose carbon monoxide (CO) is under investigation in clinical trials to treat non-cancerous diseases and has an excellent safety profile. Due to early detection and cancer awareness, an increasing number of cancer patients are diagnosed at early stages, when potentially curative surgical resection can be done. However, many patients ultimately experience recurrence. Here, we evaluate the therapeutic effect of CO on metastatic cancer progression. We show that 250 ppm CO inhibits the migration of multiple types of cancer cell lines, including breast, pancreatic, colon, prostate, liver, and lung cancer and reduces the ability to adhere to fibronectin. We demonstrate that in mouse models, 250 ppm inhaled CO inhibits lung metastasis of breast cancer and liver metastasis of pancreatic cancer. Moreover, low-dose CO suppresses recurrence and increases survival after surgical removal of primary pancreatic cancer in mice. Mechanistically, low-dose CO blocks transcription of heme importers, leading to diminished intracellular heme levels and a heme-regulated enzyme, cytochrome P4501B1 (CYP1B1). Either supplementing heme or overexpressing CYP1B1 reverses the anti-migration effect of low-dose CO. Taken together, low-dose CO therapy inhibits cell migration, reduces adhesion to fibronectin, prevents disseminated cancer cells from expanding into gross metastases, and improves survival in pre-clinical mouse models of metastasis.

Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). METHODS: To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. RESULTS: Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. CONCLUSIONS: These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Chimeric antigen receptor-T cells with cytokine neutralizing capacity.

Infusion of T lymphocytes expressing chimeric antigen receptors (CARs) can produce extraordinary antitumor activity in patients with leukemia, lymphoma, and myeloma. The signaling mechanisms activating T cells and provoking tumor cell killing also trigger cytokine secretion and macrophage activation, leading to cytokine release syndrome (CRS). CRS is a serious side effect of CAR-T cells, and proinflammatory interleukin-6 (IL-6) is central to its pathogenesis. To endow T cells with anti-CRS activity, we designed a nonsignaling membrane-bound IL-6 receptor (mbaIL6) constituted by a single chain variable fragment derived from an anti-IL-6 antibody linked to a transmembrane anchoring peptide. We found that mbaIL6 expressed on the surface of T cells could rapidly remove IL-6 from the culture supernatant. IL-6 removal was proportional to the number of mbaIL6+ cells, increased with T-cell proliferation, and neutralized IL-6 signaling and function. A construct encoding for mbaIL6 and an anti-CD19-41BB-CD3ζ CAR allowed simultaneous expression of both receptors. T cells with mbaIL6 and CAR neutralized macrophage-derived IL-6 while exerting powerful antitumor activity. Cytotoxicity and proliferation were identical to those of cells expressing CAR alone in vitro, and CAR-T cells were effective in xenograft models regardless of mbaIL6 expression. Levels of human IL-6 in mice, however, were greatly reduced if T cells expressed both receptors instead of CAR alone. Thus, CAR-T cells with on-board capacity to extinguish IL-6 represent a new approach to prevent CRS and suppress its severity without affecting the antitumor potential of CAR-T cells.

Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2 that is characterized by renal tubular epithelial cell proliferation and progressive CKD. Although the molecular mechanisms involved in cystogenesis are not established, concurrent inactivating constitutional and somatic mutations in ADPKD genes in cyst epithelium have been proposed as a cellular recessive mechanism. METHODS: We characterized, by whole-exome sequencing (WES) and long-range PCR techniques, the somatic mutations in PKD1 and PKD2 genes in renal epithelial cells from 83 kidney cysts obtained from nine patients with ADPKD, for whom a constitutional mutation in PKD1 or PKD2 was identified. RESULTS: Complete sequencing data by long-range PCR and WES was available for 63 and 65 cysts, respectively. Private somatic mutations of PKD1 or PKD2 were identified in all patients and in 90% of the cysts analyzed; 90% of these mutations were truncating, splice site, or in-frame variations predicted to be pathogenic mutations. No trans-heterozygous mutations of PKD1 or PKD2 genes were identified. Copy number changes of PKD1 ranging from 151 bp to 28 kb were observed in 12% of the cysts. WES also identified significant mutations in 53 non-PKD1/2 genes, including other ciliopathy genes and cancer-related genes. CONCLUSIONS: These findings support a cellular recessive mechanism for cyst formation in ADPKD caused primarily by inactivating constitutional and somatic mutations of PKD1 or PKD2 in kidney cyst epithelium. The potential interactions of these genes with other ciliopathy- and cancer-related genes to influence ADPKD severity merits further evaluation.

Bisphosphonates for Paget's disease of bone in adults.

BACKGROUND: Bisphosphonates are considered to be the treatment of choice for people with Paget's disease of bone. However, the effects of bisphosphonates on patient-centred outcomes have not been extensively studied. There are insufficient data to determine whether reducing and maintaining biochemical markers of bone turnover to within the normal range improves quality of life and reduces the risk of complications. OBJECTIVES: To assess the benefits and harms of bisphosphonates for adult patients with Paget's disease of bone. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, ISI Web of Knowledge and trials registers up to March 2017. We searched regulatory agency published information for rare adverse events. SELECTION CRITERIA: Randomised controlled trials (RCTs) of bisphosphonates as treatment for Paget's disease in adults. DATA COLLECTION AND ANALYSIS: Two review authors independently screened search results, extracted data and assessed studies for risk of bias. We used standard methodological procedures expected by The Cochrane Collaboration. MAIN RESULTS: We included 20 trials (25 reports, 3168 participants). Of these, 10 trials (801 participants) compared bisphosphonates (etidronate, tiludronate, ibandronate, pamidronate, olpadronate, alendronate, risedronate, zoledronate) versus placebo, seven compared two bisphosphonates (992 participants), one trial compared a bisphosphonates with a bisphosphonate plus calcitonin (44 participants), and two studies, the largest trial (1331 participants) and its interventional extension study (502 participants), compared symptomatic treatment and intensive treatment where the goal was to normalise alkaline phosphatase.Most studies were assessed at low or unclear risk of bias. Six of 10 studies comparing bisphosphonates versus placebo were assessed at high risk of bias, mainly around incomplete outcome data and selective outcome reporting.Participant populations were reasonably homogeneous in terms of age (mean age 66 to 74 years) and sex (51% to 74% male). Most studies included participants who had elevated alkaline phosphatase levels whether or not bone pain was present. Mean follow-up was six months.Bisphosphonates versus placeboBisphosphonates tripled the proportion (31% versus 9%) of participants whose bone pain disappeared (RR 3.42, 95% confidence interval (CI) 1.31 to 8.90; 2 studies, 205 participants; NNT 5, 95% CI 1 to 31; moderate-quality evidence). This result is clinically important. Data were consistent when pain change was measured as any reduction (RR 1.97, 95% CI 1.29 to 3.01; 7 studies, 481 participants).There was uncertainty about differences in incident fractures: 1.4% fractures occurred in the bisphosphonates group and none in the placebo group (RR 0.89, 95% CI 0.18 to 4.31; 4 studies, 356 participants; very low-quality evidence).None of the studies reported data on orthopaedic surgery, quality of life or hearing thresholds.Results regarding adverse effects and treatment discontinuation were uncertain. There was a 64% risk of mild gastrointestinal adverse events in intervention group participants and 48% in the control group (RR 1.32, 95% CI 0.91 to 1.92; 6 studies, 376 participants; low-quality evidence). The likelihood of study participants discontinuing due to adverse effects was slightly higher in intervention group participants (4.4%) than the control group (4.1%) (RR 1.01, 95% CI 0.41 to 2.52; 6 studies, 517 participants; low-quality evidence). Zoledronate was associated with an increased risk of transient fever or fatigue (RR 2.57, 95% CI 1.21 to 5.44; 1 study, 176 participants; moderate-quality evidence).Bisphosphonates versus active comparatorMore participants reported pain relief with zoledronate than pamidronate (RR 1.30, 95% CI 1.10 to 1.53; 1 study, 89 participants; NNT 5, 95% CI 3 to 11) or risedronate (RR 1.36, 95% CI 1.06 to 1.74; 1 study, 347 participants; NNT 7, 95% CI 4 to 24; very low quality evidence). This result is clinically important.There was insufficient evidence to confirm or exclude differences in adverse effects of bisphosphonates (RR 1.05, 95% CI 0.95 to 1.76; 2 studies, 437 participants; low-quality evidence) and treatment discontinuation (2 studies, 437 participants) (RR 2.04, 95% CI 0.43 to 9.59; 2 studies, 437 participants; very low-quality evidence).Intensive versus symptomatic treatmentThere was no consistent evidence of difference to response in bone pain, bodily pain or quality of life in participants who received intensive versus symptomatic treatment.Inconclusive results were observed regarding fractures and orthopaedic procedures for intensive versus symptomatic treatment (intensive treatment for fracture: RR 1.84, 95% CI 0.76 to 4.44; absolute risk 8.1% versus 5.2%; orthopaedic procedures: RR 1.58, 95% CI 0.80 to 3.11; absolute risk 5.6% versus 3.0%; 1 study, 502 participants; low-quality evidence).There was insufficient evidence to confirm or exclude an important difference in adverse effects between intensive and symptomatic treatment (RR 1.05, 95% CI 0.79 to 1.41; low-quality evidence).There was insufficient evidence to confirm or exclude an important difference of risk of rare adverse events (including osteonecrosis of the jaw) from the regulatory agencies databases. AUTHORS' CONCLUSIONS: We found moderate-quality evidence that bisphosphonates improved pain in people with Paget's disease of bone when compared with placebo. We are uncertain about the results of head-to-head studies investigating bisphosphonates. We found insufficient evidence of benefit in terms of pain or quality of life from intensive treatment. Information about adverse effects was limited, but serious side effects were rare, and rate of withdrawals due to side effects was low.

Long-Term Randomized Trial of Intensive Versus Symptomatic Management in Paget's Disease of Bone: The PRISM-EZ Study.

It has been suggested that normalization of bone turnover may improve clinical outcome in Paget's disease of bone (PDB) by preventing complications such as fractures and the development of osteoarthritis. Here we investigated the long-term effects of a treatment strategy that aimed to normalize bone turnover in PDB with that of symptomatic treatment. The study group comprised 502 subjects who were enrolled into a 3-year extension of the Paget's Disease: Randomized Trial of Intensive versus Symptomatic Management (PRISM) study. Intensive bisphosphonate therapy was continued in 270 of these subjects with the aim of normalizing bone turnover using zoledronic acid as the treatment of first choice. Symptomatic treatment continued in 232 subjects in whom bisphosphonates were only given for the treatment of bone pain. The primary outcome was fracture and secondary outcomes were orthopedic procedures, quality of life, and bone pain, adjusted for baseline characteristics. Serum total alkaline phosphatase (ALP) concentrations were significantly lower in the intensive group on entry to the study and the differences between groups increased as the study progressed. There were no clinically important differences in quality of life measures or bone pain between the treatment groups. Intensive treatment was associated with a nonsignificant increase in fracture risk (hazard ratio = 1.90; 95% CI, 0.91 to 3.98; p = 0.087), orthopedic procedures (1.81; 95% CI, 0.71 to 4.61; p = 0.214), and serious adverse events (relative risk 1.28; 95% CI, 0.96 to 1.42). We conclude that long-term intensive bisphosphonate therapy confers no clinical benefit over symptomatic therapy and is associated with a nonsignificant increase in the risk of fractures, orthopedic events, and serious adverse events. The results of this study suggest that in patients with established PDB, bisphosphonate therapy should focus on control of symptoms rather than suppression of bone turnover. © 2016 American Society for Bone and Mineral Research.

Pancreatic Cysts in Autosomal Dominant Polycystic Kidney Disease: Prevalence and Association with PKD2 Gene Mutations.

Purpose To define the magnetic resonance (MR) imaging prevalence of pancreatic cysts in a cohort of patients with autosomal dominant polycystic kidney disease (ADPKD) compared with a control group without ADPKD that was matched for age, sex, and renal function. Materials and Methods In this HIPAA-compliant, institutional review board-approved study, all patients with ADPKD provided informed consent; for control subjects, informed consent was waived. Patients with ADPKD (n = 110) with mutations identified in PKD1 or PKD2 and control subjects without ADPKD or known pancreatic disease (n = 110) who were matched for age, sex, estimated glomerular filtration rate, and date of MR imaging examination were evaluated for pancreatic cysts by using axial and coronal single-shot fast spin-echo T2-weighted images obtained at 1.5 T. Total kidney volume and liver volume were measured. Univariate and multivariable logistic regression analyses were conducted to evaluate potential associations between collected variables and presence of pancreatic cysts among patients with ADPKD. The number, size, location, and imaging characteristics of the cysts were recorded. Results Patients with ADPKD were significantly more likely than control subjects to have at least one pancreatic cyst (40 of 110 patients [36%] vs 25 of 110 control subjects [23%]; P = .027). In a univariate analysis, pancreatic cysts were more prevalent in patients with ADPKD with mutations in PKD2 than in PKD1 (21 of 34 patients [62%] vs 19 of 76 patients [25%]; P = .0002). In a multivariable logistic regression model, PKD2 mutation locus was significantly associated with the presence of pancreatic cysts (P = .0004) and with liver volume (P = .038). Patients with ADPKD and a pancreatic cyst were 5.9 times more likely to have a PKD2 mutation than a PKD1 mutation after adjusting for age, race, sex, estimated glomerular filtration rate, liver volume, and total kidney volume. Conclusion Pancreatic cysts were more prevalent in patients with ADPKD with PKD2 mutation than in control subjects or patients with PKD1 mutation. (©) RSNA, 2016 Online supplemental material is available for this article.

Papillary renal cell carcinoma with a somatic mutation in MET in a patient with autosomal dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 and is characterized by proliferation of renal tubular epithelium and progressive chronic kidney disease. Derangements in similar cellular signaling pathways occur in ADPKD and renal malignancies, although an association of these disorders has not been established. Herein, we present a case of papillary RCC (pRCC) incidentally discovered in a patient with ADPKD following bilateral native nephrectomy during renal transplantation. Whole exome sequencing of the pRCC found a somatic missense mutation in MET proto-oncogene, p.Val1110Ile, not present in kidney cyst epithelium or non-cystic tissue. RNA sequencing demonstrated increased mRNA expression of MET and pathway-related genes, but no significant copy number variation of MET was detected. Genetic analysis of PKD genes from peripheral blood lymphocytes and renal cyst epithelium identified a constitutional PKD1 germline mutation, p.Trp1582Ser, predicted to be pathogenic. Unique somatic mutations in PKD1 were also detected in 80% of the renal cysts analyzed, but not in the pRCC. These results suggest that, in this patient, the pRCC utilized a signaling pathway involving MET that was distinct from the pathogenesis of ADPKD. This is the first report of PKD1 mutations and a somatic mutation of the MET oncogene in a pRCC in ADPKD.

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care.

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care.

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response.

IMPORTANCE: Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. OBJECTIVE: To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. DESIGN, SETTING, AND PATIENTS: Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. MAIN OUTCOMES AND MEASURES: Feasibility, use of WES for decision making, and identification of novel biomarkers. RESULTS: A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. CONCLUSIONS AND RELEVANCE: The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Triplane fractures of the distal tibia in children.

PURPOSE: To review 28 cases of paediatric triplane fractures of the distal tibia over a 7-year period in a tertiary paediatric hospital. METHODS: Records of 21 boys and 7 girls aged 11 to 15 (mean, 13) years presented with triplane fractures of the left (n=14) and right (n=14) ankles were retrospectively reviewed. RESULTS: Injury mechanism included low-energy trauma (n=10) and moderate-energy trauma (n=18). Fractures were 2-part in 20 patients, 3-part in 6, and 4-part in 2. 11 patients had concomitant fibular fractures. Articular displacement was <2 mm in 17 patients and ≥2 mm in 11 patients. 17 patients were treated conservatively (cast immobilisation); 11 were treated surgically by closed reduction and percutaneous pinning (n=1), open reduction and percutaneous pinning (n=5), closed reduction and internal fixation (n=3), or open reduction and internal fixation (n=2). Transepiphyseal partially threaded cannulated screws were used for fixation. The mean period of casting was 5.8 weeks, and the mean non- weight bearing period was 6.1 weeks. The mean follow-up period was 14.2 (range, 9-20) weeks. All patients had excellent outcome. No patient had any infections, delayed or non-union. Six patients had decreased range of motion and 4 had pain 4 weeks after cast removal. CONCLUSION: Treatment outcome of triplane fractures of the distal tibia in children is good when guidelines are closely adhered to.

TRAF1-C5 as a risk locus for rheumatoid arthritis--a genomewide study.

BACKGROUND: Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis. METHODS: We genotyped 317,503 single-nucleotide polymorphisms (SNPs) in a combined case-control study of 1522 case subjects with rheumatoid arthritis and 1850 matched control subjects. The patients were seropositive for autoantibodies against cyclic citrullinated peptide (CCP). We obtained samples from two data sets, the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Results from NARAC and EIRA for 297,086 SNPs that passed quality-control filters were combined with the use of Cochran-Mantel-Haenszel stratified analysis. SNPs showing a significant association with disease (P<1x10(-8)) were genotyped in an independent set of case subjects with anti-CCP-positive rheumatoid arthritis (485 from NARAC and 512 from EIRA) and in control subjects (1282 from NARAC and 495 from EIRA). RESULTS: We observed associations between disease and variants in the major-histocompatibility-complex locus, in PTPN22, and in a SNP (rs3761847) on chromosome 9 for all samples tested, the latter with an odds ratio of 1.32 (95% confidence interval, 1.23 to 1.42; P=4x10(-14)). The SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5). CONCLUSIONS: A common genetic variant at the TRAF1-C5 locus on chromosome 9 is associated with an increased risk of anti-CCP-positive rheumatoid arthritis.