Electrophysiology-based stratification of pancreatic tumorigenicity by label-free single-cell impedance cytometry.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer lacking specific biomarkers that can be correlated to disease onset, promotion and progression. To assess whether tumor cell electrophysiology may serve as a marker for PDAC tumorigenicity, we use multi-frequency impedance cytometry at high throughput (∼350 cells/s) to measure the electrical phenotype of single PDAC tumor cells from xenografts, which are derived from primary pancreatic tumors versus those from liver metastases of different patients. A novel phase contrast metric based on variations in the high and low frequency impedance phase responses that is related to electrophysiology of the cell interior is found to be systematically altered as a function of tumorigenicity. PDAC cells of higher tumorigenicity exhibited lowered interior conductivity and enhanced permittivity, which is validated by the dielectrophoresis on the respective cell types. Using genetic analysis, we suggest the role of dysregulated Na+ transport and removal of Ca2+ ions from the cytoplasm on key oncogenic KRAS-driven processes that may be responsible for lowering of the interior cell conductivity. We envision that impedance cytometry can serve as a tool to quantify phenotypic heterogeneity for rapidly stratifying tumorigenicity. It can also aid in protocols for dielectrophoretic isolation of cells with a particular phenotype for prognostic studies on patient survival and to tailor therapy selection to specific patients.

Trends in use of lymphadenectomy in surgery with curative intent for intrahepatic cholangiocarcinoma.

BACKGROUND: The role of routine lymph node dissection (LND) in the surgical treatment of intrahepatic cholangiocarcinoma (ICC) remains controversial. The objective of this study was to investigate the trends of LND use in the surgical treatment of ICC. METHODS: Patients undergoing curative intent resection for ICC in 2000-2015 were identified from an international multi-institutional database. Use of lymphadenectomy was evaluated over time and by geographical region (West versus East); LND use and final nodal status were analysed relative to AJCC T categories. RESULTS: Among the 1084 patients identified, half (535, 49·4 per cent) underwent concomitant hepatic resection and LND. Between 2000 and 2015, the proportion of patients undergoing LND for ICC nearly doubled: 44·4 per cent in 2000 versus 81·5 per cent in 2015 (P < 0·001). Use of LND increased over time among both Eastern and Western centres. The odds of LND was associated with the time period of surgery and the extent of the tumour/T status (referent T1a: OR 2·43 for T2, P = 0·001; OR 2·13 for T3, P = 0·016). Among the 535 patients who had LND, lymph node metastasis (LNM) was noted in 209 (39·1 per cent). Specifically, the incidence of LNM was 24 per cent in T1a disease, 22 per cent in T1b, 42·9 per cent in T2, 48 per cent in T3 and 66 per cent in T4 (P < 0·001). AJCC T3 and T4 categories, harvesting of six or more lymph nodes, and presence of satellite lesions were independently associated with LNM. CONCLUSION: The rate of LNM was high across all T categories, with one in five patients with T1 disease having nodal metastasis. The trend in increased use of LND suggests a growing adoption of AJCC recommendations in the treatment of ICC.

Early versus late recurrence of intrahepatic cholangiocarcinoma after resection with curative intent.

BACKGROUND: The objective of this study was to investigate the characteristics, treatment and prognosis of early versus late recurrence of intrahepatic cholangiocarcinoma (ICC) after hepatic resection. METHODS: Patients who underwent resection with curative intent for ICC were identified from a multi-institutional database. Data on clinicopathological characteristics, initial operative details, timing and sites of recurrence, recurrence management and long-term outcomes were analysed. RESULTS: A total of 933 patients were included. With a median follow-up of 22 months, 685 patients (73·4 per cent) experienced recurrence of ICC; 406 of these (59·3 per cent) developed only intrahepatic disease recurrence. The optimal cutoff value to differentiate early (540 patients, 78·8 per cent) versus late (145, 21·2 per cent) recurrence was defined as 24 months. Patients with early recurrence had extrahepatic disease more often (44·1 per cent versus 28·3 per cent in those with late recurrence; P < 0·001), whereas late recurrence was more often only intrahepatic (71·7 per cent versus 55·9 per cent for early recurrence; P < 0·001). From time of recurrence, overall survival was worse among patients who had early versus late recurrence (median 10 versus 18 months respectively; P = 0·029). In multivariable analysis, tumour characteristics including tumour size, number of lesions and satellite lesions were associated with an increased risk of early intrahepatic recurrence. In contrast, only the presence of liver cirrhosis was independently associated with an increased likelihood of late intrahepatic recurrence (hazard ratio 1·99, 95 per cent c.i. 1·11 to 3·56; P = 0·019). CONCLUSION: Early and late recurrence after curative resection for ICC are associated with different risk factors and prognosis. Data on the timing of recurrence may inform decisions about the degree of postoperative surveillance, as well as help counsel patients with regard to their risk of recurrence.

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide.

AIMS: The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results. METHODS AND RESULTS: Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number. CONCLUSIONS: EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods--intercalating DNA of dead bacteria by EMA--looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.

The "mini brain" appearance of plasmacytoma in the appendicular skeleton.

We report on the case of a 70-year-old woman presenting with right hip pain. Radiographs of the right hip demonstrated a well-defined large lytic lesion in the proximal right femur, with prominent trabeculae situated peripherally and extending into the lesion in a "spoke-wheel" pattern. Magnetic resonance imaging (MRI) demonstrated solid enhancing marrow-replacing lesion, with intervening linear nonenhancing areas of low T2 signal intensity. The MRI appearance resembled that of a small brain or "mini brain". Biopsy specimen demonstrated predominantly mature plasma cells, with occasional admixed immature forms. A diagnosis of plasmacytosis, consistent with myeloma, was made. This case illustrates a rare but seemingly characteristic "mini brain" appearance of plasmacytoma, which, to date, has only been reported in the spine and has not been observed in other bony lesions.

Glenoid labrum ossification and mechanical restriction of joint motion: extraosseous manifestations of melorheostosis.

We report a case of a 47-year-old man who presented with progressive loss of motion and pain in the right shoulder. Radiographs of the shoulder demonstrated dense ossification in the glenoid and humeral head with extension into the periarticular soft tissues. CT and MRI scans confirmed the radiographic findings and also revealed ossification of the glenoid labrum. A radiographic diagnosis of melorheostosis, an uncommon benign sclerosing bone dysplasia, was made. Because of the patient's severe symptomatology, he underwent total shoulder arthroplasty. Histological analysis of the resected masses was consistent with melorheostosis with a few areas covered by a cartilage cap. This case illustrates several uncommon but important features of melorheostosis, including mechanical obstruction of joint motion requiring joint replacement, ossification of the glenoid labrum, and cartilage-covering portions of the intra-articular masses, not to be confused with cartilage-producing tumors.

Benign fibrous histiocytoma of the pterygopalatine fossa with intracranial extension.

A very rare case of fibrous histiocytoma arising in the pterygopalatine fossa with intracranial extension is described. Despite the histologic absence of nuclear pleomorphism, the tumor rapidly recurred after complete surgical resection. The patient, a 45 year old male, died shortly thereafter. The aggressive nature of our patient's tumor confirms previous observations that an aggressive radiographic appearance has prognostic value when dealing with skeletal and soft tissue tumors. The benefit of multimodal therapy has not been established in these rare head and neck lesions. In the subset of fibrous histiocytomas that invade bone, however adjunctive treatment with radiation and or chemotherapy may be appropriate.

Simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci in positive blood cultures by real-time PCR with two fluorescence resonance energy transfer probe sets.

A real-time PCR assay that uses two fluorescence resonance energy transfer probe sets and targets the tuf gene of staphylococci is described here. One probe set detects the Staphylococcus genus, whereas the other probe set is specific for Staphylococcus aureus. One hundred thirty-eight cultured isolates, which contained 41 isolates of staphylococci representing at least nine species, and 100 positive blood cultures that contained gram-positive cocci in clusters were tested. This assay was 100% sensitive and 100% specific for the detection of the Staphylococcus genus and of S. aureus.

Porcine small intestine submucosa as a flexor tendon graft.

An attractive strategy for tendon tissue engineering is the use of natural extracellular matrices as scaffold materials. One matrix that has been shown to promote healing and regeneration of neotissue in various applications is porcine-derived small intestinal submucosa. It was the objective of this study to investigate small intestinal submucosa for intrasynovial flexor tendon grafting in a canine model. We hypothesized that at 6 weeks small intestinal submucosa grafts would undergo host cell infiltration, neovascularization, and replacement by host neotendon. We also hypothesized that small intestinal submucosa grafts would be incorporated by the host without extensive adhesions to surrounding tissues and therefore maintain normal digit function. An intrasynovial tendon autograft was used as a gold standard. At 6 weeks the intrasynovial tendon autografts remained viable, contained normal numbers of cells along their length, and had minimal peritendinous adhesions. Four of six autografts had normal function as determined by rotation of the distal interphalangeal joint. Also at 6 weeks, the small intestinal submucosa grafts had host cell infiltration, neovascularization, and wavy, oriented tissue. However, ubiquitous adhesions together with impaired function in all cases suggest that small intestinal submucosa grafts in the configuration used are not suitable as full-length intrasynovial grafts in this tendon and animal model.

Bone graft incorporation in radiographically successful human intervertebral body fusion cages.

STUDY DESIGN: Biopsies were obtained from within radiographically successful human intervertebral body fusion cages to document the histology of remodeling bone graft. OBJECTIVES: The purpose of this study is to describe the tissue within successful human interbody cages with special reference to the viability of bone and the presence or absence of debris particles. SUMMARY OF BACKGROUND DATA: The use of interbody fusion cages is gaining rapid acceptance, but there is little histologic documentation of the nature of tissue within successful human interbody fusion cages. METHODS: Needle biopsies were obtained of tissue within radiographically successful intervertebral body fusion cages at the time of pedicle screw removal for back pain or fusion of adjacent spinal level in nine spinal levels of eight patients. Preoperative diagnoses of these eight adult patients included disease conditions in the sagittal plane: spondylosis (5), degenerative disc disease (6), failed laminectomy and discectomy (2), radiculopathy (1), and spondylolisthesis (1). In all cases the cages had been packed with autograft (iliac crest 7, local 1) at the time of insertion. Cage implantation was performed with anterior (anterior lumbar interbody fusion 4, corpectomy and plate fixation 1), and posterior (posterior lumbar interbody fusion 4), segmental instrumentation (plate 1, or pedicle screws 8). All cases except one cervical case had posterolateral fusion or bilateral facet fusion. The cages were composed of carbon fiber-reinforced polymer (Brantigan cage; DePuy AcroMed, Raynham, MA, n = 5) or titanium mesh (Harms Cage; DePuy AcroMed, Raynham, MA, n = 4). Cages had been in situ from 8 to 72 months (mean 28 months). All nine biopsies from eight patients were obtained from within the center of the cages. Specimens were decalcified, routinely embedded in paraffin, stained with hematoxylin and eosin, and viewed qualitatively with transmitted and polarized light. RESULTS: All needle biopsies were obtained from within the center of the cages, and no patient developed spinal instability after the biopsy. All nine biopsies showed small fragments of necrotic bone associated with viable bone and restoration of hematopoietic bone marrow. Numerous cement lines demarcated the edges of previous cycles of remodeling. The ratio of necrotic to viable bone varied greatly among cases. Small particles of debris were associated with four of the five carbon-fiber cages and one of the four specimens from titanium cages, but there was no visible bone resorption or inflammation. CONCLUSIONS: Autogenous bone graft was incorporated in these radiographically successful human intervertebral body fusion cages. A few debris particles were observed, but there was no histologic evidence of particle-induced bone resorption or inflammation.

Debris from failed ceramic-on-ceramic and ceramic-on-polyethylene hip prostheses.

To compare the properties of wear debris between ceramic-on-ceramic and ceramic-on-polyethylene total hip prostheses, particles were isolated and characterized from tissue biopsies obtained at revision arthroplasty or autopsy from two similar uncemented modular hip systems. Group A hips (11 patients; mean, 31 months in vivo) had titanium shells with alumina inserts, alumina femoral heads, and titanium alloy stems. Group B hips (seven patients; mean, 42 months) were the same as Group A but with polyethylene acetabular inserts. Particles were characterized using an electrical resistance particle analyzer, scanning electron microscope, and energy dispersive xray spectroscope. Most of the particles in Group A were ceramic, whereas most of the particles in Group B were polyethylene. Metal particles from the femoral stem and the acetabular shell also were present. If one Group A hip with impingement is excluded, the rate of particle production is significantly lower in the ceramic-on-ceramic group than in the ceramic-on-polyethylene group. With the number of samples available, no significant difference in average size could be detected among the different types of particles or among the groups.

Preliminary report of photodynamic therapy for intraperitoneal sarcomatosis.

INTRODUCTION: Sarcomatosis is the disseminated intraperitoneal spread of sarcoma. It is a condition for which there is no effective treatment. Photodynamic therapy (PDT) is a cancer treatment modality that uses a photosensitizing agent and laser light to kill cells. We report our preliminary Phase II clinical trial experience using PDT for the treatment of intraperitoneal sarcomatosis. METHODS: From May 1997 to December 1998 eleven patients received twelve PDT treatments for intraperitoneal sarcomatosis. Photofrin (PF) 2.5 mg/kg was administered intravenously 48 hours before surgical debulking to a maximum residual tumor size of less than 5 mm. Light therapy was administered at a fluence of 2.5 J/cm2 of 532 nm green light to the mesentery and serosa of the small bowel and colon; 5 J/cm2 of 630 nm red light to the stomach and duodenum; 7.5 J/cm2 of red light to the surface of the liver, spleen, and diaphragms; and 10 J/cm2 of red light to the retroperitoneal gutters and pelvis. Light fluence was measured with an on-line light dosimetry system. Response to treatment was evaluated by abdominal CT scan at 3 and 6 months, diagnostic laparoscopy at 3 to 6 months, and clinical examination every 3 months. RESULTS: Adequate tumor debulking required an omentectomy in eight patients (73%), small bowel resection in seven patients (64%), colon resection in four patients (36%), splenectomy in one patient (9%), and a left spermatic cord resection in one patient. Five patients (45%) have no evidence of disease at follow-up (range, 1.7-17.3 months), including patients at 13.8 and 17.3 months examined by CT. Two patients (18%) died from disease progression. Four patients (36%) are alive with disease progression. Toxicities related to PDT included substantial postoperative fluid shifts with volume overload, transient thrombocytopenia, and elevated liver function tests. One patient suffered a postoperative pulmonary embolism complicated by adult respiratory distress syndrome (ARDS). CONCLUSIONS: Debulking surgery with intraperitoneal PDT for sarcomatosis is feasible. Preliminary response data suggest prolonged relapse-free survival in some patients. Additional follow-up with more patients will be necessary for full evaluation of the added benefit of PDT and aggressive surgical debulking in these patients.

Paratibial cyst associated with wear debris after total knee arthroplasty.

We present a case in which a synovial cyst arose from the proximal tibia and expanded in the calf of a patient after total knee arthroplasty. A cystogram showed a direct communication between the joint cavity and the cyst, apparently associated with a screw that penetrated the tibial cortex. Histologic examination of the cyst showed an inflammatory reaction, including macrophages, foreign body giant cells, and metal and polyethylene particles. To our knowledge, this is the first case report illustrating a paraosseous cyst that developed after total knee arthroplasty. Wear debris from the total knee prosthesis may have been responsible for this unusual cyst.

Phase II trial of debulking surgery and photodynamic therapy for disseminated intraperitoneal tumors.

BACKGROUND: Photodynamic therapy (PDT) combines photosensitizer drug, oxygen, and laser light to kill tumor cells on surfaces. This is the initial report of our phase II trial, designed to evaluate the effectiveness of surgical debulking and PDT in carcinomatosis and sarcomatosis. METHODS: Fifty-six patients were enrolled between April 1997 and January 2000. Patients were given Photofrin (2.5 mg/kg) intravenously 2 days before tumor-debulking surgery. Laser light was delivered to all peritoneal surfaces. Patients were followed with CT scans and laparoscopy to evaluate responses to treatment. RESULTS: Forty-two patients were adequately debulked at surgery; these comprise the treatment group. There were 14 GI malignancies, 12 ovarian cancers and 15 sarcomas. Actuarial median survival was 21 months. Median time to recurrence was 3 months (range, 1-21 months). The most common serious toxicities were anemia (38%), liver function test (LFT) abnormalities (26%), and gastrointestinal toxicities (19%), and one patient died. CONCLUSIONS: Photofrin PDT for carcinomatosis has been successfully administered to 42 patients, with acceptable toxicity. The median survival of 21 months exceeds our expectations; however, the relative contribution of surgical resection versus PDT is unknown. Deficiencies in photosensitizer delivery, tissue oxygenation, or laser light distribution leading to recurrences may be addressed through the future use of new photosensitizers.

Wear debris from two different alumina-on-alumina total hip arthroplasties.

We compared wear particles from two different designs of total hip arthroplasty with polycrystalline alumina-ceramic bearings of different production periods (group 1, before ISO 6474: group 2, according to ISO 6474). The neocapsules and interfacial connective tissue membranes were retrieved after mean implantation times of 131 months and 38 months, respectively. Specimen blocks were freed from embedding media, either methylmethacrylate or paraffin and digested in concentrated nitric acid. Particles were then counted and their sizes and composition determined by SEM and energy-dispersive x-ray analysis (EDXA). The mean numbers and sizes of most alumina wear particles did not differ for both production periods, but the larger sizes of particle in group 1 point to more severe surface destruction. The increased metal wear in group 2 was apparently due to alumina-induced abrasion of the stems. In this study the concentrations of particles in the periprosthetic tissues were 2 to 22 times lower than those observed previously with polyethylene and alumina/polyethylene wear couples.

Open reduction and augmentation of internal fixation with an injectable skeletal cement for the treatment of complex calcaneal fractures.

OBJECTIVES: To describe the surgical handling, potential complications, and remodeling of an injectable, osteoconductive calcium phosphate cement (Norian SRS) for joint depression-type calcaneal fractures in humans, and to illustrate the clinical efficacy of this cement with special reference to early postoperative full weight bearing. DESIGN: Prospective cohort study. SETTING: Level I trauma centers in Bochum and Leipzig, Germany. INTERVENTION: Thirty-six joint depression type calcaneal fractures in thirty-two patients were augmented with the calcium phosphate cement after standard open reduction with internal fixation. Postoperative full weight bearing was allowed progressively earlier, and as the study progressed, the last patients were bearing full weight as early as three weeks postoperatively. Biopsies for histologic analysis were performed at time of hardware removal after one year (seven biopsies) or in case of infection at time of debridement (five biopsies). MAIN OUTCOME MEASURES: Clinical outcome was evaluated according to a calcaneal scoring system. Data were compared and statistically analyzed between patients with postoperative full weight bearing after eight to twelve weeks and three to six weeks, respectively. Histologic findings are described. RESULTS: Cement injection averaged ten cubic centimeters and could easily be performed under fluoroscopic control. Progressively earlier full weight-bearing was achieved without loss of reduction. There was no statistical difference in clinical outcome scores in patients with full weight bearing before or after six weeks postoperatively. The infection rate was 11 percent, possibly related to the skin incisions. The biopsies from clinically satisfactory cases showed nearly complete bone apposition, areas of vascular penetration, and reversal lines illustrating progressive cycles of resorption and new bone formation. Biopsy specimens from infected cases showed bone and cement surrounded by either fibrous tissue or acute inflammation without extensive bone apposition. CONCLUSIONS: Calcium phosphate cement augmentation of standard open reduction with internal fixation in joint-depression type calcaneal fractures allows postoperative full weight bearing as early as three weeks postoperatively. The injectable bone cement can easily be handled surgically under fluoroscopic control and has proved to be remodelable.

The spatial location of impingement in total hip arthroplasty.

Impingement between acetabular and femoral components produces wear debris and results in abnormal loads on the edge of the implant. To characterize further the spatial location of impingement and the design and alignment factors associated with impingement, we reviewed 111 retrieved acetabular components from a single manufacturer. The location of impingement in the pelvis was determined by combining the location of impingement in the retrieved implants and the spatial orientation of the acetabular components measured from available radiographs. Evidence of impingement was identified in 39% of the retrieved implants and involved the posterior portion of the acetabulum in all cases. Posterior impingement was probably the result of femoral extension and external rotation, a motion that occurs during the toe-off phase of the gait cycle. Cups with impingement were more anteverted than those without impingement (P = .016). There was a significant inverse association between impingement and the size of the femoral head, and the mean head-to-neck diameter ratio for implants with impingement was smaller than that for implants without impingement (P < .0001). Factors that appear to be associated with impingement include i) excessive cup anteversion combined with posterior positioning of the extended rim and ii) femoral components with relatively small head-to-neck diameter ratio.