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Importance: Ninety-day mortality rates after esophagectomy are an indicator of the quality of surgical oncologic management. Accurate risk prediction based on large data sets may aid patients and surgeons in making informed decisions. Objective: To develop and validate a risk prediction model of death within 90 days after esophagectomy for cancer using the International Esodata Study Group (IESG) database, the largest existing prospective, multicenter cohort reporting standardized postoperative outcomes. Design, Setting, and Participants: In this diagnostic/prognostic study, we performed a retrospective analysis of patients from 39 institutions in 19 countries between January 1, 2015, and December 31, 2019. Patients with esophageal cancer were randomly assigned to development and validation cohorts. A scoring system that predicted death within 90 days based on logistic regression ß coefficients was conducted. A final prognostic score was determined and categorized into homogeneous risk groups that predicted death within 90 days. Calibration and discrimination tests were assessed between cohorts. Exposures: Esophageal resection for cancer of the esophagus and gastroesophageal junction. Main Outcomes and Measures: All-cause postoperative 90-day mortality. Results: A total of 8403 patients (mean [SD] age, 63.6 [9.0] years; 6641 [79.0%] male) were included. The 30-day mortality rate was 2.0% (n = 164), and the 90-day mortality rate was 4.2% (n = 353). Development (n = 4172) and validation (n = 4231) cohorts were randomly assigned. The multiple logistic regression model identified 10 weighted point variables factored into the prognostic score: age, sex, body mass index, performance status, myocardial infarction, connective tissue disease, peripheral vascular disease, liver disease, neoadjuvant treatment, and hospital volume. The prognostic scores were categorized into 5 risk groups: very low risk (score, =1; 90-day mortality, 1.8%), low risk (score, 0; 90-day mortality, 3.0%), medium risk (score, -1 to -2; 90-day mortality, 5.8%), high risk (score, -3 to -4: 90-day mortality, 8.9%), and very high risk (score, =-5; 90-day mortality, 18.2%). The model was supported by nonsignificance in the Hosmer-Lemeshow test. The discrimination (area under the receiver operating characteristic curve) was 0.68 (95% CI, 0.64-0.72) in the development cohort and 0.64 (95% CI, 0.60-0.69) in the validation cohort. Conclusions and Relevance: In this study, on the basis of preoperative variables, the IESG risk prediction model allowed stratification of an individual patient's risk of death within 90 days after esophagectomy. These data suggest that this model can help in the decision-making process when esophageal cancer surgery is being considered and in informed consent.

Glioblastoma (GBM), the deadliest form of brain cancer, presents long-standing problems due to its localization. Chimeric antigen receptor (CAR) T cell immunotherapy has emerged as a powerful strategy to treat cancer. IL-13-receptor-α2 (IL13Rα2), present in over 75% of GBMs, has been recognized as an attractive candidate for anti-glioblastoma therapy. Here, we propose a novel multidisciplinary approach to target brain tumors using a combination of fluorescent, therapeutic nanoparticles and CAR T cells modified with a targeted-quadruple-mutant of IL13 (TQM-13) shown to have high binding affinity to IL13Rα2-expressing glioblastoma cells with low off-target toxicity. Azide-alkyne cycloaddition conjugation of nanoparticles to the surface of T cells allowed a facile, selective, and high-yielding clicking of the nanoparticles. Nanoparticles clicked onto T cells were retained for at least 8 days showing that the linkage is stable and promising a suitable time window for in vivo delivery. T cells clicked with doxorubicin-loaded nanoparticles showed a higher cytotoxic effect in vitro compared to bare T cells. In vitro and in vivo T cells expressing TQM-13 served as delivery shuttles for nanoparticles and significantly increased the number of nanoparticles reaching brain tumors compared to nanoparticles alone. This work represents a new platform to allow the delivery of therapeutic nanoparticles and T cells to solid tumors., Graphical abstract TQM-13 CAR T cell clicked with BPLP-PLA nanoparticles through pH-sensitive linker.Image 1, Highlights • Presenting a new strategy to combat the significant challenges in delivering nanoparticles or drugs to glioblastoma. • Designing a clickable TQM-13 CAR T cell-mediated nanoparticle drug delivery system for glioblastoma targeting and delivery. • Showcasing a CTNDDS that can be instrumental and impactful for nanomedicine, drug delivery, and cancer management.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that harbor a high potential for metastasis and have a devastating prognosis. Combination chemoradiation aids in tumor control and decreases tumor recurrence but causes deleterious side effects and does not extend long-term survival. An effective treatment with limited toxicity and enhanced efficacy is critical for patients suffering from MPNSTs.METHODSThe authors recently identified that interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed on MPNSTs and could serve as a precision-based target for delivery of chemotherapeutic agents. In the work reported here, a recombinant fusion molecule consisting of a mutant human IL-13 targeting moiety and a point mutant variant of Pseudomonas exotoxin A (IL-13.E13 K-PE4E) was utilized to treat MPNST in vitro in cell culture and in an in vivo murine model.RESULTSIL-13.E13 K-PE4E had a potent cytotoxic effect on MPNST cells in vitro. Furthermore, intratumoral administration of IL-13.E13 K-PE4E to orthotopically implanted MPNSTs decreased tumor burden 6-fold and 11-fold in late-stage and early-stage MPNST models, respectively. IL-13.E13 K-PE4E treatment also increased survival by 23 days in the early-stage MPNST model.CONCLUSIONSThe current MPNST treatment paradigm consists of 3 prongs: surgery, chemotherapy, and radiation, none of which, either singly or in combination, are curative or extend survival to a clinically meaningful degree. The results presented here provide the possibility of intratumoral therapy with a potent and highly tumor-specific cytotoxin as a fourth treatment prong with the potential to yield improved outcomes in patients with MPNSTs.

Objectives Surgical drainage is the traditional mainstay of treatment of paediatric deep neck space infection (DNSI), but recently non-operative management in selected cases has been advocated. We sought to identify any characteristics predictive of requirement for surgical intervention. Design Retrospective cohort study using receiver operating characteristics analyses. Setting Tertiary referral paediatric hospital. Participants Children (≤16 years) with a radiologically confirmed diagnosis of retro- or parapharyngeal abscess over a ten-year period. Main outcome measures Predictive value of clinical and radiological variables in determining the requirement for surgical intervention. Length of hospital stay (LoS) was also examined. Results Ninety-three children were studied, 15 (16.1%) of whom underwent immediate surgery, 42 (45.2%) of whom underwent delayed surgery following an initial period of conservative management, and 36 (38.7%) of whom were managed conservatively. Age, WCC and CRP were not predictive of the need for surgical drainage. Radiological abscess diameter, however, was predictive of requirement surgery (AUC = 0.85 [95% CI ± 0.09] P = 0.02), with a cut-off value of 2.5 cm determined assuming equal weight to sensitivity and specificity. All DNSIs were successfully treated with no adverse outcomes, and there was no significant difference in LoS between groups. In those managed surgically, outcome and LoS did not depend on yield of pus. Conclusions Selected paediatric DNSIs can be successfully managed conservatively, with abscess diameter >2.5 cm a significant predictor of need for surgical intervention. Any benefit of surgery does not appear to depend on intra-operative yield of pus.

The limited effectiveness of current therapeutics against malignant brain gliomas has led to an urgent need for development of new formulations against these tumors. Chelator Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) presents a promising candidate to defeat gliomas due to its exceptional anti-tumor activity and its unique ability to overcome multidrug resistance. The goal of this study is to develop a targeted nano-carrier for Dp44mT delivery to glioma tumors and to assess its therapeutic efficacy in vitro and in vivo. Dp44mT is loaded into poly(ethylene glycol) (PEG)ylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) decorated with glioma-targeting ligand Interlukin 13 (IL13). IL13-conjugation enhanced the NP uptake by glioma cells and also improved their transport across an in vitro blood-brain-barrier (BBB) model. This targeted formulation showed an outstanding toxicity towards glioma cell lines and patient-derived stem cells in vitro, with IC50 values less than 125 nM, and caused no significant death in healthy brain microvascular endothelial cells. In vivo, when tested on a xenograft mouse model, IL13-conjugated Dp44mT-NPs reduced the glioma tumor growth by ≈62% while their untargeted counterparts reduced the tumor growth by only ≈16%. Notably, this formulation does not cause any significant weight loss or kidney/liver toxicity in mice, demonstrating its great therapeutic potential.

Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. Despite advances in imaging technology, distinguishing between pseudoprogression and pseudoresponse remains problematic and there is a dire need for additional diagnostic methods. Tumor-derived extracellular vesicles are found in a variety of biofluids and are good candidates for a liquid biopsy. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78-96% of patients while virtually undetectable in normal brain tissue. We hypothesize that biofluids of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. We have measured the levels of IL13Rα2 in the EV fraction of plasma from GBM patients and correlated these levels to patient survival, and patient clinical characteristics (n=35). We have demonstrated that the EV fraction of some patients with GBM is enriched for the IL13Rα2 receptor. The plasma level of IL13Rα2 could also predict the level of IL13Rα2 in the GBM tumor (n=9). Further, IL13Rα2 positive EVs from patient-derived GBM cell lines could be immunoprecipitated for further interrogation of genomic and proteomic molecular markers. These exciting findings lay the groundwork for a GBM liquid biopsy based on tumor-specific EVs in patient biofluids.

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. Statement of Significance Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease.

Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells.

Key Clinical Message Acute liver dysfunction in the perioperative period may increase the risk of epidural hematoma in a patient with a neuraxial catheter. Coagulation testing needs to be carefully monitored in these patients. An epidural hematoma should be ruled out urgently by CT or MRI in cases of a persistent motor block.

The purpose of this study was to review the recent literature describing techniques, outcomes, and complications in pediatric regional anesthesia primarily among neonates and infants. Regional anesthetic techniques can be safely performed in anesthetized children, and is so widespread, that it has become accepted as standard of practice. Spinal anesthesia in the neonate requires a larger dose of local anesthetic than in adults, yet has a shorter duration of action. Neuraxial technique combined with sedation or light general anesthesia is commonly used for hernia repairs. In preterm and ex-preterm infants, awake regional techniques are reasonable alternatives. Neuraxial catheters can deliver local anesthetics and opioids both intraoperatively and postoperatively for effective pain control in all age groups including neonates. Ultrasound guidance has rapidly become the norm to ensure successful peripheral nerve blocks and abdominal wall blocks with the least amounts of local anesthetics. The dose of local anesthetic should be adjusted for each patient based on the recommended range. Regional anesthesia has shown to be safe and is being increasingly practiced. But safety should be the priority and adequate precautions should be taken to reduce potential complications.

The tumor microenvironment (TME) in glioblastoma presents a significant hurdle to effective immunotherapies as it consists of an immunosuppressive niche that results in inhibition of anti-tumor immunity. Macrophages comprise a large portion of the glioblastoma TME as they are prolific secretors of immunosuppressive cytokines and can comprise of up to 30% of tumor volume. In addition to their crucial role in immune function, macrophages are important players in iron regulation owing to their ability to efficiently sequester and release iron. We studied the impact of iron status in macrophage anti-tumor immune functions and phenotypic plasticity using RAW264.7 macrophages and bone-marrow-derived macrophages (BMDMs). Using RAW264.7 cells, we found that the iron status of macrophages impacts their immune function by modulating expression of the co-stimulation membrane proteins CD80 and CD86 as well as the mannose receptor CD206. Interestingly, the iron-mediated immunomodulation was dependent on the formulation of iron with nanoparticle formulations such as ferumoxytol (Fe-NP) upregulating CD80, CD86, and decreasing CD206 while ferric ammonium citrate (FAC) downregulated CD80, CD86 and upregulated CD206. We analyzed expression of inflammatory cytokines in BMDMs with multiplex cytokine analysis and found that both FAC and Fe-NP increased expression of the chemotactic signals CXCL10, CCL1, CCL3, and CCL4. Additionally, we found that iron status impacts the ability of macrophages to repolarize from an inflammatory, immune-activating phenotype into an immunosuppressive phenotype upon exposure to glioblastoma tumor-conditioned media. We used qRT-PCR to examine gene expression of the tumor immunity-related genes TNFa, IL1B, NOS2, and IL10 and found that iron loaded RAW264.7 macrophages stimulated with lipopolysaccharide resisted repolarization into an immunosuppressive phenotype 24 hours after exposure to glioblastoma tumor-conditioned media. Our results suggest that understanding the link between iron status and immune function in the tumor microenvironment may be an important step in improving therapies against glioblastoma.

Purpose Different socio-demographic variables, such as age, gender, and systemic disease, may affect satisfaction with complete dentures. Several studies have failed to show strong correlations either between patient satisfaction with their dentures and their quality or between denture satisfaction and the quality of the denture-supporting tissues. Hence, this study utilized a standardized questionnaire that included questions from domains such as mastication, appearance, speech, comfort, health, denture care, and social status. These questionnaires were used to determine the level of complete denture satisfaction along with socio-demographic variables such as age, gender, and systemic condition. Materials and method A total number of 128 completely edentulous patients aged between 40 and 50 years were selected. A standardized questionnaire, with 19 questions based on denture satisfaction level and masticatory capacity in the domains of functional limitation (FL), psychological discomfort (D1), psychological disability (D2), and social disability (D3), was administered. All the questions were recorded on a scale of 2, 1, 0 based on satisfied, moderately satisfied, and not satisfied, whereas hardly ever, occasionally, and very often were used for masticatory capacity. Questions on denture satisfaction were asked based on the post-treatment satisfaction with the new maxillary/mandibular complete dentures of the patients. Results Based on gender, the distribution of samples was 46.09% for male patients and 53.91% for female patients among the 128 patients selected. Similarly, based on systemic diseases, 66.41% had the presence of systemic disease while 28.13% did not have any systemic disease. Around 5.47% of the sample did not have any medical records. The predominance of psychological satisfaction was more for female patients. Based on systemic disease, it was observed that patients with the presence of systemic disease (Pn) were more psychologically comfortable than those who did not have systemic disease. The predominance of functional satisfaction was not marked in both genders. Based on systemic disease, it was observed that patients with the presence of systemic disease (Pn) had less functional comfort on mastication than those who did not have a systemic disease. Conclusion The acceptance of and satisfaction with complete denture treatment were comparatively higher in patients who had a systemic disease than in those with a non-systemic disease in terms of psychological and social comfort, whereas, in functionality, patients with a non-systemic disease had a higher satisfaction level.

Objective To assess the prevalence and determinants of childhood diseases which are covered under IMNCI in an urban resettlement colony in Delhi. Materials and methods A community-based cross-sectional study was carried out in Gokulpuri area in Delhi from January to December 2014 among 550 mothers/caretakers who were residing in the study area and selected by stratified random sampling. Data were collected using a pre-tested semi-structured questionnaire consisting of items on socio-demographic details and history of acute respiratory infections (ARI), diarrhoea, measles and malaria in the past 15 days. Data were analyzed by using SPSS 17. Chi-square test/Fisher's exact test was used for testing any significance of association and "p" value Results The study showed that 282 (51.2%) children were 1–5 years old and 290 (52.7%) were male. Prevalence of ARI was found to be 16% followed by diarrhoea to be 4.2% in the last 15 days. There was no case of measles in the area. 123 (22.3%) were found to be moderately malnourished. Prevalence of ARI was significantly less in exclusively breastfed children compared to not exclusively breastfed children (p=0.002). For other childhood diseases, the difference was not statistically significant. Conclusion The childhood diseases had a considerable burden in the area. Protective factors like colostrum and exclusive breastfeeding should be promoted by raising awareness among caregivers. Intensive health education campaigns should be carried out in the area to prevent childhood diseases.

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. The standard of care for GBM is surgical excision followed by radiation with concurrent and adjuvant temozolomide-centered chemotherapy (TMZ). Despite advances in imaging technology, early identification of treatment failure or impending resistance remains problematic. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78–96% of patients while virtually undetectable in normal brain tissue. We hypothesize that the blood of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. To detect IL13Rα2 as a specific molecular target for diagnostic testing, we have developed a modified version of IL-13 with increased affinity and specificity for IL13Rα2, and conjugated this ligand to fluorescent/ magnetic nanoparticles using an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) linker. Furthermore, we demonstrated that the EV plasma fraction of patients with GBM is enriched for the IL13Rα2 receptor. We also demonstrated the ability of the ligand-bound nanoparticles to aggregate with IL13Rα2-positive cells and EVs in vitro. This ligand-based affinity purification particle has the potential to both quantify IL13Rα2 EV levels in patient blood and isolate these EVs for further interrogation of genomic and proteomic molecular markers.

In Glioblastoma (GBM), the tumor microenvironment is critical to cancer development and progression. Glioblastoma Extracellular Vesicles (GBM-EVs) have been shown to promote a tumor-supporting phenotype in macrophages, leading to surrounding immunosuppression and enhanced tumor growth. Microglia and macrophages contribute to iron homeostasis, essential for cellular energy production in rapidly dividing cells. The HFE mutation is associated with altered cellular iron homeostasis. It is the most common autosomal recessive polymorphism found in Caucasians and may be linked to worse patient prognosis in GBM. Our lab showed that HFE mutation confers phenotypic differences between HFE and wild-type (WT) macrophages, with HFE macrophages displaying increased migration and phagocytosis. How these phenotypic differences may present in response to GBM exosomes has not yet been explored. Primary macrophage cultures isolated from bone marrow of WT and HFE mutant knock-in mice were exposed to exosomes from different patient-derived cancer stem cells. Significant differences in macrophage viability 24 hours after exosome exposure were observed in response to GBM exosomes (p= 0.001). When exposed to GBM exosomes, WT macrophage viability consistently decreased while the viability of macrophages with the HFE mutation increased. There was no significant difference between the two genotype macrophage groups when exposed to U87 exosomes, supporting that the stem cells are modulating macrophage phenotype. These data suggest that genotypic differences in iron handling pay a critical role in macrophage response to GBM exosomes and may be responsible for differences seen in disease severity in patients with HFE mutations.

Mucormycosis is a fulminant fungal infection that occurs most often in diabetic and immunocompromised patients, including those with hematologic malignancies. Patients with maxillary resections present a challenge situation for the maxillofacial prosthodontist. Prosthodontic rehabilitation of such patients presents a significant challenge in restoring speech, deglutition and mastication, and respiration. This report discusses the impact of post-surgical management of mucormycosis patient with prosthodontic treatment and evaluating the oral health-related quality of life using the Oral Health Impact Profile-14 (OHIP-14) questionnaire.

Background Detection of glioma with MRI contrast agent is limited to cases in which the blood-brain barrier (BBB) is compromised as contrast agents cannot cross the BBB. Thus, an early-stage infiltrating tumor is not detectable. Interleukin-13 receptor alpha 2 (IL-13Rα2), which has been shown to be overexpressed in glioma, can be used as a target moiety. We hypothesized that liposomes conjugated with IL-13 and encapsulating MRI contrast agent are capable of passing through an intact BBB and producing MRI contrast with greater sensitivity. Methods The targeted MRI contrast agent was created by encapsulating Magnevist (Gd-DTPA) into liposomes conjugated with IL-13 and characterized by particle size distribution, cytotoxicity, and MRI relaxivity. MR image intensity was evaluated in the brain in normal mice post injection of Gd-DTPA and IL-13-liposome-Gd-DTPA one day apart. The specificity for glioma detection by IL-13-liposome-Gd-DTPA was demonstrated in an intracranial glioma mouse model and validated histologically. Results The average size of IL-13-liposome-Gd-DTPA was 137 ± 43 nm with relaxivity of 4.0 ± 0.4 L/mmole-s at 7 Tesla. No significant cytotoxicity was observed with MTS assay and serum chemistry in mice. The MRI signal intensity was enhanced up to 15% post injection of IL-13-liposome-Gd-DTPA in normal brain tissue following a similar time course as that for the pituitary gland outside of the BBB. MRI enhanced by IL-13-liposome-Gd-DTPA detected small tumor masses in addition to those seen with Magnevist-enhanced MRI. Conclusions IL-13-liposome-Gd-DTPA is able to pass through the uncompromised BBB and detect an early stage glioma that cannot be seen with conventional contrast-enhanced MRI.

Peripheral nerve sheath tumors are benign tumors that have the potential to transform into malignant peripheral nerve sheath tumors (MPNSTs). Interleukin-13 receptor alpha 2 (IL13Rα2) is a cancer associated receptor expressed in glioblastoma and other invasive cancers. We analyzed IL13Rα2 expression in several MPNST cell lines including the STS26T cell line, as well as in several peripheral nerve sheath tumors to utilize the IL13Rα2 receptor as a target for therapy. In our studies, we demonstrated the selective expression of IL13Rα2 in several peripheral nerve sheath tumors by immunohistochemistry (IHC) and immunoblots. We established a sciatic nerve MPNST mouse model in NIH III nude mice using a luciferase transfected STS26T MPNST cell line. Similarly, analysis of the mouse sciatic nerves after tumor induction revealed significant expression of IL13Rα2 by IHC when compared to a normal sciatic nerve. IL13 conjugated liposomal doxorubicin was formulated and shown to bind and internalized in the MPNST cell culture model demonstrating cytotoxic effect. Our subsequent in vivo investigation in the STS26T MPNST sciatic nerve tumor model indicated that IL13 conjugated liposomal doxorubicin (IL13LIPDXR) was more effective in inhibiting tumor progression compared to unconjugated liposomal doxorubicin (LIPDXR). This further supports that IL13 receptor targeted nanoliposomes is a potential approach for treating MPNSTs.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas arising from peripheral nerves. MPNSTs have increased expression of the oncogene aurora kinase A, leading to enhanced cellular proliferation. This makes them extremely aggressive with high potential for metastasis and a devastating prognosis; 5-year survival estimates range from a dismal 15% to 60%. MPNSTs are currently treated with resection (sometimes requiring limb amputation) in combination with chemoradiation, both of which demonstrate limited effectiveness. The authors present the results of immunohistochemical, in vitro, and in vivo analyses of MLN8237 for the treatment of MPNSTs in an orthoxenograft murine model.METHODSImmunohistochemistry was performed on tumor sections to confirm the increased expression of aurora kinase A. Cytotoxicity analysis was then performed on an MPNST cell line (STS26T) to assess the efficacy of MLN8237 in vitro. A murine orthoxenograft MPNST model transfected to express luciferase was then developed to assess the efficacy of aurora kinase A inhibition in the treatment of MPNSTs in vivo. Mice with confirmed tumor on in vivo imaging were divided into 3 groups: 1) controls, 2) mice treated with MLN8237, and 3) mice treated with doxorubicin/ifosfamide. Treatment was carried out for 32 days, with imaging performed at weekly intervals until postinjection day 42. Average bioluminescence among groups was compared at weekly intervals using 1-way ANOVA. A survival analysis was performed using Kaplan-Meier curves.RESULTSImmunohistochemical analysis showed robust expression of aurora kinase A in tumor cells. Cytotoxicity analysis revealed STS26T susceptibility to MLN8237 in vitro. The group receiving treatment with MLN8237 showed a statistically significant difference in tumor size compared with the control group starting at postinjection day 21 and persisting until the end of the study. The MLN8237 group also showed decreased tumor size compared with the doxorubicin/ifosfamide group at the conclusion of the study (p = 0.036). Survival analysis revealed a significantly increased median survival in the MLN8237 group (83 days) compared with both the control (64 days) and doxorubicin/ifosfamide (67 days) groups. A hazard ratio comparing the 2 treatment groups showed a decreased hazard rate in the MLN8237 group compared with the doxorubicin/ifosfamide group (HR 2.945; p = 0.0134).CONCLUSIONSThe results of this study demonstrate that MLN8237 is superior to combination treatment with doxorubicin/ifosfamide in a preclinical orthoxenograft murine model. These data have major implications for the future of MPNST research by providing a robust murine model as well as providing evidence that MLN8237 may be an effective treatment for MPNSTs.

Glioblastoma (GBM) is the most lethal and incurable brain cancer. Despite maximum treatment, high recurrence rates result in median survival of less than 2 years. A subset of treatment resistant cells within GBM known as glioma initiating cells (GICs) are implicated in recurrence and exhibit ferritin overexpression. High expression of ferritin is known to be associated with poor overall survival in GBM patients. Our previous finding showed that downregulation of the ferritin heavy chain subunit (FTH1) enhanced treatment sensitivity in astrocytoma cells. Recently, functional importance of FTH1 in GICs was demonstrated by shRNA silencing leading to ablation of tumorigenic ability. We therefore posited that disrupting iron metabolism through FTH1 knockdown would radiosensitize GICs. Thus we developed multivalent cationic liposomes that enabled delivery of FTH1 siRNA to patient derived GICs isolated from pro-neural (PN) and mesenchymal (Mes) GBM. In both subtypes, FTH1 loss led to increased apoptosis and reduced cell viability. When exposed to radiation, PN GICs were unable to repair and recover from DNA damage, leading to a further decrease in cell viability and a complete loss of reproductive clonogenic potential unlike their more radioresistant Mes counterparts. Moreover, expression of stem cell marker Nestin was markedly reduced in PN but not Mes GICs. Thus, FTH1 loss is detrimental to both GIC subtypes, it seems to be critical for maintaining stemness and radiation resistance of PN GICs. These findings further support the viability of FTH1 as a therapeutic target in GBM.

Elevated expression of the iron regulatory protein, ferritin heavy chain 1 (FTH1), is increasingly being associated with high tumor grade and poor survival outcomes in glioblastoma. Glioma initiating cells (GICs), a small population of stem-like cells implicated in therapeutic resistance and glioblastoma recurrence, have recently been shown to exhibit increased FTH1 expression. We previously demonstrated that FTH1 knockdown enhanced therapeutic sensitivity in an astrocytoma cell line. Therefore, in this study we developed a liposomal formulation to enable the in vitro delivery of FTH1 siRNA in patient xenograft derived GICs from glioblastomas with pro-neural and mesenchymal transcriptional signatures to interrogate the effect of FTH1 downregulation on their radiation sensitivity. Transfection with siRNA decreased FTH1 expression significantly in both GICs. However, there were inherent differences in transfectability between pro-neural and mesenchymal tumor derived GICs, leading us to modify siRNA: liposome ratios for comparable transfection. Moreover, loss of FTH1 expression resulted in increased extracellular lactate dehydrogenase activity, executioner caspase 3/7 induction, substantial mitochondrial damage, diminished mitochondrial mass and reduced cell viability. However, only GICs from pro-neural glioblastoma showed marked increase in radiosensitivity upon FTH1 downregulation demonstrated by decreased cell viability, impaired DNA repair and reduced colony formation subsequent to radiation. In addition, the stemness marker Nestin was downregulated upon FTH1 silencing only in GICs of pro-neural but not mesenchymal origin. Using liposomes as a siRNA delivery system, we established FTH1 as a critical factor for survival in both GIC subtypes as well as a regulator of radioresistance and stemness in pro-neural tumor derived GICs. Our study provides further evidence to support the role of FTH1 as a promising target in glioblastoma.

Background: Since periodontal diseases are primarily inflammatory in nature, the ability to detect inflammatory lesions in gingival tissue is essential for the diagnosis and monitoring of changes in gingival status. Gingival bleeding has been used as a reliable and objective clinical parameter not only for the evaluation of inflammatory conditions of periodontium, but also in the detection of its activity and progression. The gingival bleeding index (Loe) is based on the presence or absence of gingival bleeding, thereby provides a means of converting observed clinical data into statistical analysis. Periodontitis is widely regarded as a bacterial infection mainly mediated by subgingival plaque. N-benzoyl-DL-arginine β-naphthylamide (BANA) test is a simple chair side test used to detect, the specific enzymatic activity of microorganisms involved in periodontal disease. Objective: The objective of this study is to compare the clinical evaluation of the gingival index (GI) with the enzymatic BANA test. Materials and Methods: A total of 320 sites were selected from 20 patients with generalized chronic periodontitis, based on the presence are absence of gingival bleeding according to GI of Loe. Sub-gingival plaque samples were collected using a sterile curette and the BANA test was performed. Results: The distribution of GI and the intensity of the BANA test for the 320 sites analyzed during the initial examination of patients, with significance determined by the Chi-square test. The results were statistically significant at P ≤ 0.000. Conclusion: There was a direct relationship between positive BANA test results and GI scores, clearly demonstrating the validity of gingival bleeding as a significant diagnostic parameter. Clinical Significance: The validity of gingival bleeding as a clinical tool not only for the diagnosis of periodontal disease, but also in the detection of its activity and progression could be established by correlating the GI scores (which is based on the presence or absence of bleeding) with the primary etiologic factors (putative periodontal pathogens) assessed by BANA assay.

Background We propose that the symptoms associated with paraesophageal hernia (PEH) are more diverse than previously suggested, and symptoms and clinical manifestations correlate to the anatomy of the hernia. Methods Patients undergoing surgery for PEH were reviewed from a prospective, institutional review board–approved, single-center database. Presenting symptoms, anatomy of the PEH, demographics, and outcomes were analyzed from 2000 to 2010. Presenting symptoms were assessed for incidence and improvement after surgery. Size and configuration of the PEH were assessed with respect to presenting symptoms. Results The study included 270 consecutive patients, 63% were female, and the median age was 70 years (range, 39 to 94 years). The most common presenting symptoms were heartburn in 175 patients (65%), early satiety in 136 patients (50%), chest pain in 130 patients (48%), dyspnea in 130 patients (48%), dysphagia in 129 patients (48%), regurgitation in 128 patients (47%), and anemia in 112 patients (41%). Two hundred sixty-nine patients (99.6%) had at least one symptom; the median number of symptoms was 4 (range, 0 to 10). The type of PEH was II (n = 10), III (n = 206), and IV (n = 54), and the percent intrathoracic stomach was less than 50% (n = 33), 50% to 74% (n = 86), 75% to 99% (n = 55), and 100% (n = 96). Paraesophageal hernia type was significantly associated with heartburn (type II/III; p = 0.005) and dyspnea (type IV; p = 0.007). Significant associations included lower percent intrathoracic stomach with regurgitation ( p = 0.04); higher percent intrathoracic stomach with early satiety ( p = 0.02), decreased meal size ( p = 0.007), and dyspnea ( p p = 0.001). With a median postoperative follow-up of 103 days, symptoms were subjectively better in patients with dyspnea (67%), early satiety (79%), regurgitation (92%), dysphagia (81%), chest pain (76%), and heartburn (93%). Conclusions Paraesophageal hernia is associated with a greater diversity of symptomatic presentation than previously thought. Asymptomatic patients are rare, and size and configuration of the hernia are associated with specific symptoms. Patients with large PEHs should be assessed by an experienced surgeon for elective repair.

ObjectiveAssessment of the clinical impact of giant paraesophageal hernias have historically focused on upper gastrointestinal symptoms. This study assesses the effect of paraesophageal hernia repair on respiratory function.MethodsAll patients undergoing repair of giant paraesophageal hernia were prospectively entered into a database approved by the institutional review board. Patients had symptoms documented preoperatively, including dyspnea. Pulmonary function tests (PFTs) were done preoperatively and repeated a median of 106 days after repair (range, 16-660 days).ResultsPreoperative and postoperative PFTs were obtained in 120 unselected patients treated for paraesophageal hernia between 2000 and 2010. Patients’ median age was 74 years (range, 45-91 years), 74 (62%) were female, and median body mass index was 28.0 (range, 16.8-46.6). Median length of stay was 4 days (range, 3-10 days), and perioperative mortality was zero. Hernias were classified as type II in 3 (3%) patients, III in 92 (77%), and IV in 25 (21%). Percent of intrathoracic stomach was assigned from preoperative contrast studies and grouped as less than 50% (n = 6; 5%), 50% to 74% (n = 35; 29%), 75% to 99% (n = 29; 24%), and 100% (n = 50; 42%). Preoperative symptoms included heartburn 71 (59%), early satiety 65 (54%), dyspnea 63 (52%), chest pain 48 (40%), dysphagia 56 (47%), regurgitation 47 (39%), and anemia 44 (37%). PFTs significantly improved after paraesophageal hernia repair (mean volume change, percent reference change): forced vital capacity +0.30 L,+10.3%pred; FEV1 +0.23 L,+10.4%pred (all P

Approximately half of all gliomas are resistant to chemotherapy, and new therapeutic strategies are urgently needed to treat this cancer. We hypothesized that disrupting iron homeostasis in glioma cells could block tumor growth, based on an acute requirement for high levels of iron to meet energy requirements associated with their rapid growth. Ferritin is best known as an intracellular iron storage protein, but it also localizes to tumor cell nuclei where it seems to protect DNA from oxidative damage and to promote transcription. In this study, we hypothesize that silencing the H-ferritin (heavy chain ferritin) gene could increase tumor sensitivity to chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to several human cancer cell lines by using cationic liposomes (C-liposome). H-ferritin siRNA decreased protein expression by 80% within 48 hours, and this decrease was associated with more than 50% decrease in the LD50 for DNA-alkylating agent carmustine (BCNU), which is commonly used to treat glioma in clinic. In a subcutaneous mouse model of human glioma, intratumoral injections of liposomes containing H-ferritin siRNA reduced the effective dose of BCNU needed for tumor suppression by more than 50%. A plasmid supercoil relaxation assay showed that H-ferritin specifically and directly protected DNA from BCNU treatment. H-ferritin siRNA additionally seemed to increase apoptosis in glioma cells in vitro upon H-ferritin knockdown. Overall, our results illustrate how silencing H-ferritin can effectively sensitize tumors to chemotherapy and also show the ability of C-liposomes to serve as a novel in vivo delivery tool for siRNAs. Cancer Res; 71(6); 2240–9. ©2011 AACR.

Objective Outcomes assessing various treatment modalities for esophageal cancer primarily report results in terms of morbidity, mortality, survival, and quality of life. The most appropriate stage-by-stage treatment for esophageal cancer remains controversial. There are limited data outlining the comparative costs of surgical, combined modality and definitive chemoradiation treatments, and added costs associated with complications. Methods Between 2000 and 2004, 4 treatment groups were studied: surgery alone, chemotherapy followed by surgery, chemoradiotherapy followed by surgery, and chemoradiotherapy alone. Fifteen consecutive patients from each group receiving their entire treatment at Virginia Mason Medical Center were identified. Patient demographics and outcomes were taken from a prospective institutional review board-approved surgical database, and chart review obtained information for neoadjuvant therapy and definitive chemoradiotherapy groups. Treatment-related costs were extracted from Virginia Mason Medical Center’s financial data management system between date of diagnosis to 90 days after completion of primary therapy. Results Treatment groups were similar in age, gender ratio, American Society of Anesthesiologists status, body mass index, and tumor cell type. Costs increased with the number of treatment modalities: surgery alone, $33,517; chemotherapy followed by surgery, $41,875; chemoradiotherapy followed by surgery, $47,389; and chemoradiotherapy alone, $46,659. Treatment-related complications were surgery alone, 47%; chemotherapy followed by surgery, 64%; chemoradiotherapy followed by surgery, 66%; and chemoradiotherapy alone, 87% ( P = . 139). Complications increased costs in all groups: surgery alone, 26% ( P = . 008); chemotherapy followed by surgery, 23% ( P = . 001); chemoradiotherapy followed by surgery, 9% ( P = .702); and chemoradiotherapy alone, 19% ( P = .248). Conclusions Costs vary significantly among treatment approaches: surgery alone costs 80% of chemotherapy and surgery, 71% of chemoradiotherapy and surgery, and 72% of chemoradiotherapy alone. Costs of tri-modality therapy and definitive chemoradiotherapy are similar. Especially in the absence of definitive evidence-based data, these costs should be a factor in the production of future national treatment guidelines. Decreasing costs requires future quality initiatives in esophageal cancer treatment that focus on minimizing complications related to treatment.

Glioblastoma (GBM) is the most common primary central nervous system (CNS) tumor in the United States and yet despite aggressive treatment, the median survival remains at 8 months. About 75% of human glioblastoma tumors selectively overexpress the α(2) subtype of interleukin 13 (IL-13Rα(2)), considered a “decoy receptor” for GBM, allowing it to evade apoptosis normally induced by IL-13 binding. Our laboratory and others have developed targeting strategies to take advantage of IL-13Rα(2) expression on GBMs. We showed that wildtype IL-13 targeted nanoliposomes delivering doxorubicin (WT-IL13-Dox), resulted in over 5-fold reduction of orthotopic tumors, with 60% of mice surviving for >200 days compared to mice treated with unconjugated liposomes. WT-IL13, however, also binds to the shared IL-13/IL-4 receptor α(1) subunit found in multiple organs, including the heart and lungs, suggesting potential cross-reactivity with surrounding tissues. To address off-target effects, a mutant version of IL-13 was developed known as Targeted Quadruple Mutant-13 (TQM), which display improved binding affinity to the IL-13Rα(2) receptor, and decreased affinity to the IL-13/IL-4 receptor α(1) subunit. Twenty mice were normalized by tumor burden into two groups, one treated with WT-IL13-Dox and the other group with TQM-13-conjugated liposomal doxorubicin (TQM-13-Lip-Dox). Each group received 5mg/kg of doxorubicin in the liposomes for 4 weeks. Mice treated with TQM-targeted liposomes had slower tumor growth, smaller tumor burden, and prolonged survival (33 vs 23 days, p=0.009) than those treated with WT-IL13 liposomes. WBC counts in WT-IL13 mice suggested they were immunocompromised in comparison to TQM mice (p=0.02), which may contribute to worse survival. These findings suggest that TQM-13 bound nanoliposomes may enable the development of targeted therapy with a decreased side effect profile for delivery of chemotherapeutic agents to GBMs.

Glioblastoma is the most common central nervous system malignancy in adults with a median survival of only 14 months. The current arsenal of treatment modalities including surgical resection and combination chemoradiation have been largely ineffective. One reason proposed for the ineffectiveness of our current therapeutic regimen is the resistance of glioma stem cells (GSCs) within the tumor and tumor borders. We investigated the hypothesis that the communication of GSCs to their microenvironment through exosomes is a key factor underlying the enhanced cellular proliferation and the development of resistance to therapeutics. Exosomes are nanometer sized vesicles released by cancer cells that contain DNA, RNA, and protein critical to the interaction of a cell with its microenvironment. Two properties of exosomes were analyzed: 1) exosome function and 2) exosome profile. Exosomes secreted by patient derived-glioma stem cells (GSC-exosomes) increased cellular proliferation, radiation resistance, temozolomide resistance, and doxorubicin resistance. We further profiled the GSC-exosomes to begin to probe the underlying mechanism of this phenomenon. Profiling showed specific changes to RNA and protein favoring therapeutic resistance and cellular proliferation. For example, GSC exosomes have increased expression of proteins involved in radiation and chemotherapeutic resistance (Ex. CDK4 and Notch), cellular proliferation (Ex. Cyclin B1 and Cyclin D2), angiogenesis (Ex. VEGF-A and EGFR), glioma cell stemness and de-differentiation (Ex. EPHA2, Cathepsin B), and cell invasion and metastasis (Ex. ITGA3, COL4A2). The results of our study suggest a novel exosome-based mechanism by which glioma stem cells alter therapeutic resistance and increase cellular proliferation. TUMOR MODELS

Doxorubicin is a topoisomerase II inhibitor demonstrated to be effective in several cancers including malignant peripheral nerve sheath tumors (MPNST) and glioblastoma in cell culture, but at higher dose causes adverse effects like cardiotoxicity in patients. This minimizes the therapeutic dosage of doxorubicin administered for such cancers. To overcome this issue we have developed novel lipid based nanovesicles carrying doxorubicin with certain chemosensitizing agents like farnesyl thiosalicylic acid (FTS), a Ras inhibitor and dexrazoxane (DEX) in the lipid bilayer component of the nanovesicles using a microfluidic encapsulation method. FTS modified nanovesicles with doxorubicin in the core demonstrated an enhanced cytotoxic potential in glioma cells and neurofibromatosis type-1 associated MPNST cells (NF-1 MPNST). To determine the in vivo therapeutic efficacy, U87-Luc subcutaneous tumor bearing mice were injected twice weekly with 4mg/kg body weight dose of FTS-LIP-DOX or LIP-DOX (n=8) for 6 weeks. After 6 weeks of treatment, the median tumor volume in the control group reached 3212 mm(3) and that of LIP-DOX and FTS-LIP-DOX reached a mean volume of 111 mm(3) and 64.4 mm(3) respectively. In a second formulation, doxorubicin was combined with dexrazoxane (DEX), a metal chelator and topoisomerase inhibitor in a liposome formulation. In our pilot study in vivo investigation we treated mice with DOX-DEX-liposomes(n=3) or DOX-liposomes (n=2) at a dose of 7 mg/kg body weight with respect to doxorubicin, twice weekly for 5 weeks in a MPNST mouse model. In the first 3 weeks, a slower tumor progression in the DOX-DEX liposomes group was observed compared to DOX-liposomes formulation, beyond which the tumor progression in both groups approached each other. We didn’t observe any adverse effect based on serum chemistry analysis. This novel therapy provides an opportunity for doxorubicin dose escalation in MPNST mouse model with improved therapeutic efficacy.

262 Background: As the number of clinically actionable genetic markers for lung adenocarcinoma has increased, the need for accurate and timely identification of these markers has become paramount. In January 2018, our practice began systemically using NGS on all lung adenocarcinoma biopsy specimens. We compared NGS testing to our previous standard (PCR- and FISH-based assays) with regards to breadth of markers tested and time to diagnosis. Methods: We compared detection of mutations in EGFR, ALK, and ROS1 and time from resection or biopsy to test results in recently diagnosed patients from January-March 2018 with stage III/IV adenocarcinoma of the lung to similar patients diagnosed January-December 2016. Results: 63 patients were eligible for analysis: 17 patients in 2018 and 46 patients in 2016. Mean time to diagnosis was 13 days for EGFR, 16 days for ALK and 18 days for ROS1 in 2016, based on previous methods (PCR/FISH) compared to 27 days for EGFR, 26 days for ALK and 26 days for ROS1 in 2018 with NGS testing. Four of the 46 samples (8.6%) were found to have insufficient tissue using PCR/FISH, whereas 1 out of 17 (5.8%) samples were found to be insufficient with NGS testing. NGS testing returned up to 35 molecular markers, including those which might be targets for clinical trial therapy. Conclusions: The use of NGS testing increased the number of actionable molecular markers tested and appears to have resulted in an increase in the time taken to diagnose EGFR, ALK, and ROS1. The clinical significance of this time difference (i.e. time to treatment initiation) as well as ultimate cost of NGS compared to PCR/FISH remain to be determined. FIGURE 1: Time to diagnosis of molecular tests in lung adenocarcinoma stage III/IV using PCR/FISH from January through December 2016 and NGS from January through March 2018.[Table: see text]

Background We describe our experience with laparoscopic pancreaticoduodenectomy, including 5-year actuarial survival rates. Study Design This is a retrospective study of selected patients who underwent laparoscopic pancreaticoduodenectomy at a single center between 1998 and 2006. We have described the salient features of our technique and followup protocol. Patient characteristics, histologic variety of the tumor, resection margins, morbidity, mortality, and actuarial survival rates were studied. Results The procedure could be completed laparoscopically with tumor-free margins in all patients, including patients with ampullary carcinoma (n = 24), pancreatic cystadenocarcinoma (n = 4), pancreatic head adenocarcinoma (n = 9), low common bile duct cancer (n = 3), and two patients with chronic pancreatitis with a suspicious mass lesion in the head of pancreas. Mean age of patients was 61 years (range 28 to 70 years). There was a single perioperative mortality. Overall followup rate was 95.1%, with two patients lost to followup at 22 and 36 months. Among the survivors, two patients have metastatic disease and local recurrence developed in one patient. Five-year actuarial survival rates for all patients with malignancy, ampullary adenocarcinoma, pancreatic cystadenocarcinoma, pancreatic head adenocarcinoma, and common bile duct adenocarcinoma are 32%, 30.7%, 33.3%, 19.1%, and 50%, respectively. Presence of microscopic lymph node involvement is associated with poor survival, although operations in the setting of chronic pancreatitis resulted in increased morbidity. Conclusions Laparoscopic pancreaticoduodenectomy can be performed with safety and good results in properly selected patients. Localized malignant lesions, irrespective of histopathology, are particularly amenable to this approach.

Peripheral nerve sheath tumors are benign tumors that have the potential to transform to malignant peripheral nerve sheath tumor (MPNST). In our initial study we found that majority of the MPNST cells and tissues express IL13Rα2 receptor, which could be a potential target for therapy. We have also observed earlier that the IL13 conjugated liposomal doxorubicin was effectively endocytosed by established MPNST cells like ST88-14 and sNF96.2 and induces cytotocity. To determine the in vivo efficacy, sciatic nerve tumor mouse model was developed by injecting the luciferase transfected STS26T cells in the sciatic nerve. The tumor progression was monitored following the injection of luciferin intraperitoneally and imaging with intravital imaging spectroscopy (IVIS). All the mice developed tumor in 2 weeks after implantation of tumor cells in the sciatic nerve as evidenced by IVIS imaging. Once the tumor was visualized the mice were treated with IL-13 conjugated and non-conjugated liposomal doxorubicin for 7 weeks at a low dose of 4 mg/kg body weight weekly once. Similarly the tumor sections from the mice which were treated with liposomes were analyzed for apoptotic markers like Caspase 3. The tumor volume, weight of the mice and the serum chemistry were recorded. Although the tumor progression was controlled initially, the efficacy was reduced with increasing tumor volume. So currently we are adopting a more frequent dosage regimen instead of weekly once strategy. Furthermore, as Ras activation has also been reported to promote the Schwann cell to MPNST transformation, we combined the doxorubicin with farnesyl thiosalicylic acid (FTS, a Ras inhibitor) in the liposome formulation to study their tumor regression property in such models. In conclusion we developed a successful MPNST animal model to monitor the tumor progression by non-invasive method and currently utilizing this model for investigating the therapeutic efficacy of multifunctional nanovesicles.

TUMOR MICROENVIRONMENT TMIC-01. TUMOR TARGETED QUANTUM DOTS TO IDENTIFY GLIOMA INITIATING CELLS AND EXOSOMES Achutha Madhankumar, Oliver Mrowczynski, Suhag Patel, Elias Rizk, Cody Weston, Michael Glantz, Lichong Xu, Christopher Siedlecki, and James Connor; Pennsylvania State University College of Medicine, Hershey, PA, USA GBM and certain brain metastatic cancers possess invasiveness and high infiltrating potential making them harder to detect at the earlier stage of the disease progression. Identification of cancer stem cells and exosomes secreted by them in the biofluids like CSF will be an ideal way to determine the presence of residual cancer after therapy. We utilize ligand conjugated quantum dots (QD) to specifically label the cancer stem cells and exosomes to identify the presence of certain invasive markers. Human glioma initiating cells were cultured in adherent form and were exposed to interleukin-13 conjugated quantum dots (IL13QD), which binds the glioma initiating cells expressing IL13Ra2 receptor and also the exosomes secreted by the glioma stem cells. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Ra2 receptor. Fluorescent microscopy, electron microscopy and flow cytometry were performed on the exosomes after complexing with nano quantum dots (QD). IL13QD was demonstrated to specifically bind glioma stem cells both in the monolayer and spheroid culture. The binding affinity of the exosomes to the quantum dots were also confirmed by atomic force microscopy. The morphology and size of exosomes were confirmed by electron microscopy and dynamic light scattering method. The density plot from the flow cytometer experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that in ex vivo, tumor targeted quantum dots can bind with tumor initiating glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. There is a future possibility to differentiate the metastatic tumor associated exosomes from the non-metastatic exosomes based on their QD binding profile Neuro-Oncology 17:v221–v225, 2015. doi:10.1093/neuonc/nov236.1 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

Glioblastoma multiforme (GBM) is the most prevalent, lethal, malignant and refractory primary brain tumor with a very dismal survival rate. The glioma initiating cell (GIC) population within GBMs has been associated with treatment resistance and subsequently been implicated in tumor recurrence. Studies from our lab had previously shown that expression of the heavy chain subunit of the iron storage protein ferritin, H-ferritin, is essential for the survival and therapeutic resistance of non-stem glioma cells. Since H-ferritin is overexpressed in GICs, we hypothesized that downregulating its expression in these cells would lead to increased radiosensitivity. We therefore developed a novel strategy to sensitize these recalcitrant GICs to radiation therapy using a multivalent cationic liposome formulation that could efficiently transfect and deliver H-ferritin siRNA to GICs in vitro. Using patient derived T3691 CD133+ GICs we showed that downregulating H-ferritin led to a reduction in cell viability compared to controls upon being radiated at 4Gy and 8Gy dosage. Additionally these cells also showed an increased expression of the DNA damage response protein phospho-ϒH2AX upon radiation over controls. Thus, we have demonstrated that loss of H-ferritin in GICs leads to increased radiation sensitivity. Ongoing studies are focused on delineating the mechanisms through which H-ferritin loss mediates this effect in GICs.

Malignant peripheral nerve sheath tumor is a soft tissue sarcoma that poses tremendous challenges for effective therapy. Several therapeutic approaches using drugs such as doxorubicin and rapamycin have been tried in the past with minimum success. Therapeutic resistant property mediated by MDR proteins, in combination with DNA damage repair was proposed as a major cause leading to therapeutic inefficacy of chemotherapeutics in MPNST tumors. To overcome this issue, we encapsulated doxorubicin in lipid nanovesicles and targeted to IL13Rα2, a receptor that is selectively overexpressed in several malignant peripheral nerve sheath tumor cell lines. In the present investigation, a MPNST mouse model was developed using STS26T-Luc cell line stably transfected with luciferase reporter gene. These cells were implanted into the sciatic nerve that could be imaged by Intravital Imaging Spectroscopy (IVIS). After confirming the tumor formation, the mice were injected with 7 mg/kg body weight of (a) IL13 linked liposomal doxorubicin (IL3LIPDXR) (b) unconjugated liposomal doxorubicin (LIPDXR) and (c) control mice injected with equal volume of saline. The mice were treated weekly once for 7 weeks. The mice which received IL13LIPDXR and LIPDXR showed significant reduction in tumor volume compared to the control group. Among the targeted and non-targeted liposomes, IL13LIPDXR showed rapid tumor regression. However, beyond 4 weeks, the drug was ineffective in shrinking or maintaining the tumor volume. This concludes that although the targeted delivery of doxorubicin, seems to be effective initially however in the later course of treatment they are ineffective in controlling the tumor progression. Approaches such as combining doxorubicin with a small molecule Ras inhibitor or other agents in the liposome formulation which are currently underway may help to improve the therapeutic efficacy.

Glioblastoma Multiforme (GBM) is the most common CNS malignancy in adults, and even with multimodal therapies, carries a poor prognosis. These tumors are heterogeneous and modify their environment, promoting tumor growth, invasion, and therapeutic resistance. The intratumoral heterogeneity of GBM is accentuated during treatment. Even in the face of surgical resection and adjuvant chemoradiation, the unfortunate but inevitable recurrence and progression of GBM is nearly universal. This mechanism of acquired radiation resistance is not understood. We believe that acquired resistance of GBM is due to exosomes secreted from radiated GBM cells conferring resistance to the local cell population. Exosomes are nanometer cell-derived vesicles containing RNAs and proteins. Growing evidence demonstrates exosomes are instrumental to a cell’s interaction with its microenvironment, and have the ability to transfer information to recipient cells contributing to tumorigenicity. We utilized nanoparticle tracking analysis to analyze characteristics of exosome secretion after exposure to radiation. We used cell proliferation assays and cell survival assays to interrogate radiation derived exosome effects on the local cancer cell population. We assessed the potential of exosome blocking agents as a possible avenue for therapeutic manipulation to abrogate this oncogenic effect. We demonstrate that resistance and proliferative factors in GBM derived exosomes change after radiation exposure. Moreover, radiation derived exosomes have functional impact on tumor cell proliferation and radiation resistance. We also demonstrate the therapeutic potential of exosome blocking agents to inhibit acquired resistance, which develops in nearly all GBM patients. Radiation-derived exosomes cause increases in cellular proliferation and survival in the local cancer cell population and may be the hidden mediators of an ultimately radiation resistant tumor recurrence and the dire prognosis that follows. Adjuvant therapy with exosome blocking agents may be used to inhibit these oncogenic effects, leading to optimal patient outcomes.

High grade brain tumors like glioblastoma multiforme (GBM) and certain other brain metastatic cancers possess high tumor invasive and infiltrating properties making them harder to detect. Identification of cancer stem cells and exosomes secreted by them in the biofluids like cerebrospinal fluid (CSF) will be an ideal way of detecting the presence of residual cancer after therapy. In the current approach, a ligand conjugated quantum dot (QD) was utilized to specifically label the cancer stem cells and exosomes to identify the presence of IL13Rα2 a tumor invasive marker. Human glioma initiating cells were cultured in adherent form and were exposed to interleukin-13 conjugated quantum dots (IL13QD), which binds the glioma initiating cells expressing IL13Rα2 receptor. Similarly the exosomes isolated from cancer stem cell condition media and CSF was briefly incubated with IL13QD, before analyzing their complexing pattern. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Rα2 receptor. Fluorescent microscopy, transmission electron microscopy and flow cytometry were performed on the exosomes after complexing with IL13QD. IL13QD was demonstrated to specifically bind glioma stem cells both in the monolayer and spheroid culture. The binding affinity of the exosomes to the quantum dots were quantitatively and qualitatively confirmed by atomic force microscopy (AFM). The morphology and size of exosomes before and after complexing with exosomes were confirmed by electron microscopy and AFM. The density plot from the flow cytometry experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that tumor targeted quantum dots can selectively bind with glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. This binding profile could be utilized to identify the expression of certain tumor promoting receptors and antigens on the surface of exosomes present in the CSF and cancer stem cells from the glioma patients. Experiments are in progress to identify the tumor initiating cells in an orthotopic mouse glioma tumor model after intravenous administration of wild type IL13 and certain high affinity mutated IL13 conjugated quantum dots, by non-invasive way. Citation Format: Achuthamangalam B. Madhankumar, Oliver Mrowczynski, Brad Zacharia, Michael Glantz, Lichong Xu, Becky Webb, Christopher Siedlecki, Akiva Mintz, James R. Connor. Interleukin-13 conjugated quantum dots to identify glioma initiating cells and exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2017-745

Fibromatosis is a rare fibroproliferative disorder with a tendency for local infiltrative and destructive growth. Local recurrence is frequent, despite apparent complete resection after radical excision. We present a case of a 22-year-old woman with massive recurrent thoracic fibromatosis extending into the neck and impairing the function of her right upper limb. This required a multidisciplinary approach to surgery to salvage the limb. The case highlights the fact that while every attempt should be made to achieve negative histologic margins, local recurrence is not uncommon. Therefore, if fibromatosis occurs adjacent to or involves vital structures, these should not be sacrificed to achieve negative margins. Function and structure preserving procedures are important as the primary goal, if not even more important.

Cancer cells generally exhibit increased iron requirements and heightened iron metabolism. As the major iron storage protein, ferritin expression is elevated in many types of cancer. In this study, we report that the sensitization of H-ferritin down-regulation in glioma cells against radiation and suggest the potential of H-ferritin down-regulation as an adjuvant therapy in human glioma. In human glioma cells, down-regulation of H-ferritin performed through a nanotechnology-based transfection platform led to a transient decrease in transferrin receptor level, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn produced intracellular oxidative stress, demonstrated by protein oxidation damage, as well as a decrease in the stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance. In glioma cells, exposure to radiation resulted in protein oxidation which could be exacerbated by H-ferritin down-regulation. Previously we have demonstrated a DNA protection role for H-ferritin. Here we expand this concept by showing that the down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. The activation of DNA repair mechanisms was impaired when H-ferritin was absent. The in vitro data suggest a synergistic effect of radiation and H-ferritin down-regulation. Additionally, we expand our research into CD133-positive glioma stem cells (GSCs), which are notoriously resistant to anti-cancer treatment. Down-regulation of H-ferritin in GSCs inhibited cell proliferation in vitro, presumably through apoptosis induction. With an intracranial glioma model established by GSC implantation, we demonstrated that the survival was significantly prolonged by H-ferritin siRNA transfection through intravenous injection, in tumor bearing mice treated with first-line drug Temodar. This study supports the potential of H-ferritin siRNA as an adjuvant therapy in glioma treatment.

Glioblastoma Multiforme is an aggressive brain tumor combining high infiltrative properties with a heterogenous nature making it challenging to identify and remove them by surgery and increasing their resistance to other conventional therapies. GBM tumors are known to express the IL-13Rα2 receptors which we have used to target nanoliposomes for enhancing theranostic efficacy. In our present investigation we utilized interleukin-13 ( IL-13) conjugated quantum dots (IL-13QD) to selectively bind the glioma stem cells in culture and the soluble receptor in a medium and the exosomes released in a biological fluid. The binding phenomenon was characterized by various techniques like atomic force microscopy, dynamic light scattering method, electron microscopy and fluorescent microscopy. Investigation was also performed to determine the release of soluble IL-13Rα2 receptor and IL-13Rα2 expressing exosomes from the glioma stem cells in the culture media and in the cerebrospinal fluid. After confirming the presence of soluble receptor by immunoblots and ELISA and exosomes by electron microscopy and dynamic light scattering method, in vitro studies were performed with the IL-13QD in a media containing the exosomes and soluble receptors to investigate the binding and aggregation properties of the IL-13QD. Our investigation revealed the aggregation properties of the IL-13QD in the presence of soluble receptor and exosomes indicating a correlation between the receptor expression and aggregation and dissociation of quantum dots. Further studies are in progress with patient samples that would lead to a development of a biomarker and a therapeutic agent for the highly malignant brain tumors.

Malignant melanoma is the deadliest form among all skin cancers. Fortunately, if detected early, even malignant melanoma may be treated successfully. In this paper, a new intelligent method of classifying benign and malignant melanoma lesions is used. As the first step of the image analysis, preprocessing techniques are used to remove noise and undesired structures from the images using filter such as median filtering. Segmentation is one of the important steps in cancer automatic detection, because it can greatly affect on the results of detection. In the second step, a simple thresholding method is used to segment and localize the lesion, a boundary tracing algorithm is also implemented to validate the segmentation. In the third step, the different features are extracted from a segmented image and classified by using Stolz algorithm.

Human glioblastoma tumors selectively express receptors for interleukin 13 (IL-13). In a previous study, we showed that liposomes, when conjugated with IL-13, will deliver chemotherapeutics to a subcutaneous glioma tumor model in mice much more effectively than conventional unconjugated liposomes. Based on this observation, we developed an intracranial brain tumor model in nude mice using human U87 glioma cells. Mice receiving weekly i.p. injections of 15 mg/kg of doxorubicin encapsulated in IL-13–conjugated liposomes had a 5-fold reduction in the intracranial tumor volume over 6 weeks and four of seven animals survived >200 days after tumor implantation. In contrast, the animals receiving unconjugated liposomes with the same doxorubicin concentration did not survive beyond 35 days and there was no evidence of tumor size reduction. The presence of liposomes with doxorubicin in the tumor was shown by taking advantage of the selective expression of IL-13 receptors on the tumor cells and the endogenous fluorescence of doxorubicin. There was no increase in the indices of toxicity in animals receiving the doxorubicin-containing liposomes. Finally, a model of the blood-brain barrier was used to show that the nanovesicles do not harm the endothelial cells yet maintain their toxicity to astrocytoma cells. This approach is necessary to show the efficacy of this targeting platform for tumors in which the blood-brain barrier is not compromised and as a potential use of the nanovesicle system as a surveillance mechanism to prevent recurrence. These data show that IL-13 targeted nanovesicles are a viable option for the treatment of brain tumors. [Mol Cancer Ther 2009;8(3):648–54]

Impending paradoxical embolism through a patent foramen ovale with concomitant pulmonary embolism is a rare entity that requires urgent treatment. We present the case of a 74-year-old woman with acute pulmonary embolism and atrial flutter and with echocardiographic features of impending paradoxical embolism through the patent foramen ovale and tricuspid regurgitation. We performed an open pulmonary embolectomy with removal of thrombus from both atria, closure of the patent foramen ovale, a right-sided modified Maze procedure, and De Vega's tricuspid annuloplasty. The patient made a satisfactory recovery. Surgical management is preferable in this setting because it provides the opportunity to correct any associated cardiac abnormalities.