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The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Peroxisome proliferator-activated receptor α (PPARα) regulates fatty acid oxidation (FAO). Usually, very-long chain fatty acids are first activated by acyl-CoA synthetase (ACS) to generate acyl-CoA for oxidation by acyl-CoA oxidase (ACOX) in peroxisomes, and the resultant shorter chain fatty acids will be further oxidized in mitochondria. ACS long-chain family member 4 (ACSL4) preferentially uses arachidonic acid (AA) as substrates to synthesize arachidonoyl-CoA. Arachidonoyl-CoA is usually esterified into phospholipids. When AA is released by phospholipase A2 (PLA2) from phospholipids, it will be used for prostaglandin synthesis by cyclooxygenases (COX). In this study, when PPARα agonist WY-14,643 was mixed in liquid Lieber-DeCarli ethanol or control diets and fed to mice, liver PLA2, COX-2, and ACOX1 were induced but ACSL4 was inhibited, suggesting that AA released by PLA2 from phospholipid will be metabolized to prostaglandin via COX-2 instead of being synthesized into acyl-CoA by ACSL4. However, liver prostaglandin E2 (PGE2), a major component of prostaglandin, was not increased with the induced COX-2 but decreased by WY-14,643. ACOX1 specific inhibitor mixed in the liquid diets restored both the WY-14,643-suppressed liver TG and PGE2, but COX-2 specific inhibitor celecoxib mixed in the liquid diets reversed the WY-14,643-suppressed liver TG but not liver PGE2 contents. These results suggest that induction of PLA2, COX-2 and ACOX1 orchestrates to increase oxidation of AA/PGE2, which constitutes one new mechanism by which PPARα induces peroxisomal FAO and inhibits ethanol-induced liver fat accumulation.

Arthroplasty implants are comprised of metal alloys designed to function within the human body. Implant-related issues and associated soft-tissue reactions have been well documented for modular revision hip and knee constructs. This case highlights findings of metallosis in the context of polyethylene wear in a failed primary total knee arthroplasty. Fretting of a polyethylene reinforcement pin within the tibial baseplate as a direct result of knee joint instability appears to be the root cause of observed periprosthetic metallosis. Enhanced design principles and improved polyethylene locking mechanisms may be useful to potentially mitigate fretting-related issues in future knee replacement designs. The authors recommend surveillance in patients with this construct especially when prosthetic instability is present.

Background: Obesity affects ∼20% of children in the United States and reports of successful dietary treatment are lacking. This study aimed to determine the change in body weight in severely obese ...

We report a case of high-grade endometrial stromal sarcoma originally assumed to be a benign leiomyoma, with BCOR internal tandem duplicates resulting in BCOR overexpression. A 34-year-old female presented to the gynecologic oncology service for recent miscarriage attributed to a large leiomyoma. She reported symptoms of vaginal bleeding and abdominal pain. Myomectomy and tissue analysis was performed which revealed BCOR internal tandem duplicates and cyclin D1 positive high grade endometrial sarcoma. Definitive treatment for the sarcoma was performed with pelvic washings, robotic assisted total laparoscopic hysterectomy with bilateral salpingo-oophorectomy, omentectomy, and pelvic lymph nodes dissection. Negative margins were obtained along with negative lymph node and omental sections. Her sarcoma was staged pT1b (FIGO 1B). At follow up, patient reported no symptoms and was informed no further treatment was indicated. It is rare for high grade endometrial stromal sarcoma (HG-ESS) to present in a 34-year-old woman, though BCOR internal tandem duplicate (ITD) high grade endometrial stromal sarcoma is more common in younger women. This patient also had a unique finding in the pathology report, positive progesterone receptor (PR) expression, making this an interesting but difficult diagnosis.

Fibroblast growth factor 21 (FGF21) is mainly regulated by peroxisome proliferator-activated receptor α (PPARα) in liver. The PPARα-FGF21 axis protects against alcohol-related liver disease (ALD). FGF21 exerts its effect via FGF receptor 1 (FGFR1). However, liver specific FGFR1 abrogation had no effect on ALD. Adipose tissues highly express FGFR1. When adipocyte specific FGFR1 knockout (fgfr1(adipoQ-cre)) mice and corresponding normal control (fgfr1(fl/fl)) mice were fed with Lieber-DeCarli ethanol liquid diet for 3 weeks, liver triglyceride (TG) accumulation was increased in the fgfr1(fl/fl) mice to a greater extent than in the fgfr1(adipoQ-cre) mice. When PPARα agonist WY-14,643 was added in the liquid ethanol diet at 10 mg/L, the ethanol-induced liver TG accumulation was blunted in the fgfr1(fl/fl) mice but not in the fgfr1(adipoQ-cre) mice. There was no significant difference in WY-14,643-induced fatty acid oxidation, ethanol metabolism, and oxidative stress between the fgfr1(fl/fl) and fgfr1(adipoQ-cre) mice. Interestingly, adipose atrophy was induced by WY-14,643 in the fgfr1(adipoQ-cre) mice but not in the fgfr1(fl/fl) mice. Serum free fatty acid was also decreased by WY-14,643 in the fgfr1(adipoQ-cre) mice but not in the fgfr1(fl/fl) mice. These results suggest that WY-14,643 inhibits alcoholic fatty liver and regulates adipose tissue mass and fat mobilization from adipose tissues to liver in an adipocyte FGFR1-dependent manner.

Peroxisome proliferator-activated receptor α (PPARα), a fatty acid oxidation regulator, inhibits alcohol-induced fatty liver (AFL). PPARα agonist WY-14,643 ameliorates AFL. Nicotine enhances AFL. In this study, we investigated whether PPARα activation also blocks nicotine-enhanced AFL. Mice were fed liquid diets containing ethanol in the presence or absence of nicotine, WY-14,643 was added to the above diets at 10 mg/L. The results showed that WY-14,643 blunted AFL and nicotine-enhanced AFL, which was paralleled with striking induction of PPARα target genes. However, serum ALT was dramatically increased by the ethanol/WY-14,643 feeding and was further increased by nicotine/ethanol/WY-14,643 feeding, which was confirmed by necro-inflammation and elevated oxidative stress. Interestingly, serum alcohol levels were dramatically decreased by WY-14,643. Ethanol is mainly metabolized by alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1) and catalase. ADH and CYP2E1 were not increased by WY-14,643, but catalase was induced. What is more, injection of catalase inhibitor increased serum ethanol. Decreased serum alcohol, attenuated fatty liver, and enhanced liver injury were not induced by WY-14,643 in mice lacking PPARα. In conclusion, PPARα activation by WY-14,643 attenuates alcohol/nicotine-induced fatty liver but deteriorates ethanol/nicotine-induced liver injury; WY-14,643 enhances ethanol metabolism via induction of catalase.

A 40-year-old male with a right-sided neck mass was diagnosed with metastatic melanoma. A repeat positron-emission tomography after treatment with combination immunotherapy demonstrated increased hypermetabolic activity in the right supraclavicular, hilar, and mediastinal regions. Immunotherapy was discontinued and a BRAF/MEK inhibitor combination was started. Repeat imaging showed a decrease in size of the neck mass; however, hilar and mediastinal lymph nodes increased in size. A fine needle aspiration of mediastinal lymph nodes was consistent with a granulomatous process. A diagnosis of a sarcoid-like reaction (SLR) was made, and he was started on steroids. A follow-up positron emission tomography showed decreased hilar and mediastinal lymph node hypermetabolic activity. We, therefore, report this rare case of immunotherapy-induced SLR to the expanding literature on immunotherapy-related adverse effects and would like to highlight that SLR can occur in conjunction with disease progression making it challenging to distinguish between the two.

e17584 Background: Disease recurrence and progression of ovarian cancer is a common event, which is accompanied by the development of platinum-resistant or refractory disease. The presence of chemo-resistant cancer stem cells (CSCs) contributes to tumor propagation, maintenance, and treatment resistance of this difficult to treat disease. The choice of appropriate chemotherapy is critical to the management of advanced epithelial ovarian cancer. We developed a CSCs assay (ChemoID) that identifies the most effective chemotherapy for individual patients from a panel of FDA-approved chemotherapies. Methods: Ascites or interventional radiology biopsies were collected under physician order from 85 consecutive patients affected by 3rd relapsed ovarian cancer. Test results from the assay were used to treat patients with the highest cell kill drugs, taking into consideration their health status and using dose reductions, if needed. Our study assessed the correlation of the ChemoID test results to treatment outcomes independently of other biomarkers. A retrospective chart analysis and review of CT and PET scans were performed to determine patients’ outcomes for tumor response, progression-free survival (PFS), and overall survival (OS). Results: We observed that recurrent ovarian cancer patients treated with high-cell kill chemotherapy agents guided by the ChemoID drug response assay had an improvement in their median PFS and OS when compared to historical median PFS and OS and/or when compared to patients who could not receive high cell kill chemotherapies (PFS low cell kill 3.5 months vs. high cell kill 12.0 months; OS low cell kill 6.0 months vs. high cell kill 15.0 months). Conclusions: The current evidence strongly suggests that the ChemoID assay predicted the effectiveness of specific therapies, as well as improved outcomes for patients treated with assay-sensitive therapies.

Obesity is linked to nonalcoholic steatohepatitis. Peroxisome proliferator-activated receptor-α (PPARα) regulates lipid metabolism. Cytochrome P-450 2A5 (CYP2A5) is a potential antioxidant and CYP2A5 induction by ethanol is CYP2E1 dependent. High-fat diet (HFD)-induced obesity and steatosis are more severe in CYP2A5 knockout (cyp2a5(−/−)) mice than in wild-type mice although PPARα is elevated in cyp2a5(−/−) mice. To examine why the upregulated PPARα failed to prevent the enhanced steatosis in cyp2a5(−/−) mice, we abrogate the upregulated PPARα in cyp2a5(−/−) mice by cross-breeding cyp2a5(−/−) mice with PPARα knockout (pparα(−/−)) mice to create pparα(−/−)/cyp2a5(−/−) mice. The pparα(−/−)/cyp2a5(−/−) mice, pparα(−/−) mice, and cyp2a5(−/−) mice were fed HFD to induce steatosis. After HFD feeding, more severe steatosis was developed in pparα(−/−)/cyp2a5(−/−) mice than in pparα(−/−) mice and cyp2a5(−/−) mice. The pparα(−/−)/cyp2a5(−/−) mice and pparα(−/−)mice exhibited comparable and impaired lipid metabolism. Elevated serum alanine transaminase and liver interleukin-1β, liver inflammatory cell infiltration, and foci of hepatocellular ballooning were observed in pparα(−/−)/cyp2a5(−/−) mice but not in pparα(−/−) mice and cyp2a5(−/−) mice. In pparα(−/−)/cyp2a5(−/−) mice, although redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 and its target antioxidant genes were upregulated as a compensation, thioredoxin was suppressed, and phosphorylation of JNK and formation of nitrotyrosine adduct were increased. Liver glutathione was decreased, and lipid peroxidation was increased. Interestingly, inflammation and fibrosis were all observed within the clusters of lipid droplets, and these lipid droplet clusters were all located inside the area with CYP2E1-positive staining. These results suggest that HFD-induced fibrosis in pparα(−/−)/cyp2a5(−/−) mice is associated with steatosis, and CYP2A5 interacts with PPARα to participate in regulating steatohepatitis-associated fibrosis. NEW & NOTEWORTHY PPARα is upregulated in cyp2a5(−/−) mice, but HFD-induced steatosis is still deteriorated. PPARα abrogation makes cyp2a5(−/−) mice more sensitive to HFD-induced steatosis, liver inflammation, and fibrosis, suggesting that PPARα upregulation in cyp2a5(−/−) mice is a compensation response. HFD-induced liver inflammation, fibrosis, and nitrotyrosine formation in pparα(−/−)/cyp2a5(−/−) mice are all within clusters of lipid droplets, and lipid droplets are all within CYP2E1-positive area.

Introduction: The prognosis of glioblastoma (GBM) treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ)/radiotherapy remains very poor (less than 15 months). GBMs have been found to contain a small population of cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID) that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male), all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) associations of 12-month recurrence, PFS, and OS outcomes were estimated for CSC, bulk tumor, and combined assay responses for the standard-of-care TMZ treatment; sensitivities/specificities, areas under the curve (AUCs), and risk reclassification components were examined. Results: Median follow-up was 8 months (range 3-49 months). For every 5% increase in in vitro CSC cell kill by TMZ, 12-month patient response (nonrecurrence of cancer) increased two-fold, OR=2.2 ( P =.016). Similar but somewhat less supported associations with the bulk tumor test were seen, OR=2.75 ( P =.07) for each 5% bulk tumor cell kill by TMZ. Combining CSC and bulk tumor assay results in a single model yielded a statistically supported CSC association, OR=2.36 ( P =.036), but a much attenuated remaining bulk tumor association, OR=1.46 ( P =.472). AUCs and [sensitivity/specificity] at optimal outpoints (>40% CSC cell kill and >55% bulk tumor cell kill) were AUC=0.989 [sensitivity=100/specificity=97], 0.972 [100/89], and 0.989 [100/97] for the CSC only, bulk tumor only, and combined models, respectively. Risk categorization of patients was improved by 11% when using the CSC test in conjunction with the bulk test (risk reclassification nonevent net reclassification improvement [NRI] and overall NRI=0.111, P =.030). Median recurrence time was 20 months for patients with a positive (>40% cell kill) CSC test versus only 3 months for those with a negative CSC test, whereas median recurrence time was 13 months versus 4 months for patients with a positive (>55% cell kill) bulk test versus negative. Similar favorable results for the CSC test were observed for PFS and OS outcomes. Panel results across 14 potential other treatments indicated that 34/41 (83%) potentially more optimal alternative therapies may have been chosen using CSC results, whereas 27/41 (66%) alternative therapies may have been chosen using bulk tumor results. Conclusions: The ChemoID CSC drug response assay has the potential to increase the accuracy of bulk tumor assays to help guide individualized chemotherapy choices. GBM cancer recurrence may occur quickly if the CSC test has a low in vitro cell kill rate even if the bulk tumor test cell kill rate is high.

BACKGROUND The presence of chemotherapy-resistant cancer stem cells (CSC) leading to tumor recurrence is postulated to be one of the major factors in the poor response to the current standard of care treatments in progressive high-grade gliomas. Unfortunately, in spite of several new drug discoveries for other tumor types, not many advances have been reported for this disease and therefore recurrent and progressive high-grade glioma survival remains dismal. METHODS We have used a CLIA and CAP accredited Cancer Stem Cell Cytotoxicity Assay(ChemoID) to guide most effective chemotherapy treatments from a panel of FDA approved drugs or their combinations for 12 recurrent glioblastoma (GBM) patients and 2 progressive anaplastic gliomas, all eligible to receive a stereotactic biopsy. Patients were evaluated by MRI scans and response was assessed according to RANO criteria. RESULTS The median age of our patient cohort was 49 years (21–63), with 11 males (79%) and 3 females (21%). We observed 6 complete responses (CR) 43%, 6 partial responses (PR) 43%, and 2 progressive diseases (PD) 14%. Of note, the two PD were observed in patients that could not be treated with assay recommended therapy due to their health status. Patients treated with ChemoID assay-directed therapy had a longer median overall survival (OS) of 13.3 months (5.4-NA) compared to the historical median OS of 9.0 months (8.0–10.8 months) previously reported (1). Notably, our recurrent and progressive high-grade glioma patient cohort treated with assay-guided therapy had a 57% probability to survive at 12 months, compared to the 27% historical probability of survival at 12 months observed in previous studies (1). CONCLUSIONS The data suggests that the ChemoID Cancer Stem Cell Cytotoxicity Assay has the potential to help guide individualized chemotherapy choices to improve recurrent and progressive high-grade glioma patient outcome. 1) Friedman, H.S. et. al. JCO2009 27:28, 4733–4740.

Lung adenocarcinoma (LAC) accounts for the majority of all non-small cell lung cancer (NSCLC) cases. A substantial proportion of LAC patients present with local and distant metastasis at the time o...

Obesity increases the risk of postmenopausal breast cancer (BC). This risk is mediated by obesity-induced changes in the adipose-derived secretome (ADS). The pathogenesis of BC in obesity is stimulated by mTOR hyperactivity. In obesity, leucine might support mTOR hyperactivity. Leucine uptake by BC cells is through L-Type Amino Acid Transporter 1 (LAT1). Our objective was to link obesity-ADS induction of LAT1 to the induction of mTOR signaling. Lean- and obese-ADS were obtained from lean and obese mice, respectively. Breast ADS was obtained from BC patients. Estrogen-receptor-positive BC cells were stimulated with ADS. LAT1 activity was determined by uptake of 3H-leucine. The LAT1/CD98 complex, and mTOR signaling were assayed by Western blot. The LAT1 antagonists, BCH and JPH203, were used to inhibit LAT1. Cell migration and invasion were measured by Transwell assays. The results showed obese-ADS-induced LAT1 activity by increasing transporter affinity for leucine. Consistent with this mechanism, LAT1 and CD98 expression were unchanged. Induction of mTOR by obese-ADS was inhibited by LAT1 antagonists. Breast ADS from patients with BMIs &gt, 30 stimulated BC cell migration and invasiveness. Collectively, our findings show that obese-ADS induction of LAT1 supports mTOR hyperactivity in luminal BC cells.

Metastatic esophageal adenocarcinoma to the urinary bladder is extremely rare. We describe a previously healthy 49-year-old female with recent diagnosis of adenocarcinoma of the gastroesophageal junction with metastatic disease to the liver. Biopsy was positive for human epidermal growth factor receptor 2 (HER2) by Fluorescence In Situ Hybridization (FISH). She received six cycles of Cisplatin, 5-Fluorouracil, and Herceptin and subsequently developed symptomatic anemia and hematuria. Cystoscopy with retroflexion was performed and she received a transurethral resection of bladder tumor with fulguration. Pathology of the bladder tumor revealed similar morphology to her liver metastasis and immunohistochemical stains were consistent with metastatic esophageal cancer. Three weeks after being diagnosed with metachronous urinary bladder metastasis from esophageal adenocarcinoma primary, she expired. She only received her first cycle of palliative chemotherapy with Ramucirumab and Paclitaxel.

Tumor heterogeneity implies the possibility of significantly different expression of key pathways between primary and metastatic clones. Colon adenocarcinoma is one of the few tumors where current practice includes resection of primary and isolated organ metastases simultaneously without neoadjuvant therapy. We performed a pilot study on 28 cases of colon adenocarcinoma resected simultaneously with metastases in patients with no history of neoadjuvant therapy. We assayed matched primary and metastatic tumors from each patient with common diagnostic antibodies to Bcl-2, Cyclin D1, AMACR, and ALDH-1 by immunohistochemistry with semi-quantitative interpretation on archived formalin fixed, paraffin embedded samples. We were powered for large, consistent differences between primary and metastatic expression, and found 21 of 28 had a significant difference in expression of at least one of the four proteins, accounting for multiplicity of testing. Cyclin D1 had significantly more cases with differential metastatic:primary expression than would be expected by chance alone (p-value 0.0043), favoring higher expression in the metastatic sample. Bcl-2 and ALDH-1 had trends in this direction (p-value 0.078 each). Proportionately more cases with significant differences were identified when a liver metastasis was tested. We conclude differences in expression between metastatic and primary colon adenocarcinoma within the same patient exist, and may have therapeutic and biomarker testing consequences.

Introduction Disease recurrence and progression of ovarian cancer is common with the development of platinum-resistant or refractory disease. This is due in large part to the presence of chemo-resistant cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. We developed a CSCs drug cytotoxicity assay (ChemoID) to identify the most effective chemotherapy treatment from a panel of FDA approved chemotherapies. Methods Ascites and pleural fluid samples were collected under physician order from 45 consecutive patients affected by 3rd-5th relapsed ovarian cancer. Test results from the assay were used to treat patients with the highest cell kill drugs, taking into consideration their health status and using dose reductions, as needed. A retrospective chart review of CT and PET scans was used to determine patients' outcomes for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Results We observed that recurrent ovarian cancer patients treated with high-cell kill chemotherapy agents guided by the CSCs drug response assay had an improvement in the median PFS corresponding to 5.4 months (3rd relapse), 3.6 months (4th relapse), and 3.9 months (5th relapse) when compared to historical data. Additionally, we observed that ovarian cancer patients identified as non-responders by the CSC drug response assay had 30 times the hazard of death compared to those women that were identified as responders with respective median survivals of 6 months vs. 13 months. We also found that ChemoID treated patients on average had an incremental cost-effectiveness ratio (ICER) between -$18,421 and $7,241 per life-year saved (LYS). Conclusions This study demonstrated improved PFS and OS for recurrent ovarian cancer patients treated with assay-guided chemotherapies while decreasing the cost of treatment., Highlights • Ovarian cancer progression and recurrence is mostly attributed to the presence of cancer stem cells (CSCs), which are chemo-resistant. • Eliminating CSCs is a strategy that could improve patients' outcome. • We developed a CSC drug cytotoxicity assay to identify the most effective chemotherapy treatment from a panel of FDA approved chemotherapies. • Recurrent ovarian cancer patients treated with high-cell kill chemotherapy agents guided by the CSCs drug response assay had an improvement in the median PFS when compared to historical data.

Background In the United States endometrial carcinoma is the most common female gynecologic malignancy. An average of more than 60,000 new cases of endometrial carcinomas have been diagnosed yearly over the past 5 years, with a higher incidence occurring in the central Appalachian states of Ohio and West Virginia. In the U.S., the national average of newly diagnosed endometrial carcinomas is 26.8 in every 100,000 women, while in the states of Ohio and West Virginia the average is 30.5 and 31.1 in every 100,000 women, respectively. This notable increase in the incidence of endometrial carcinomas may be due a variety of elevated risk factors including but not limited to: tobacco use, obesity, and genetic predisposition of the predominant demographic. The American Cancer Society estimates that approximately 55,000 new cases of endometrial carcinoma will be diagnosed in 2020 yet, this disease is widely considered understudied and under-represented in mainstream cancer research circles. Methods The aim of this study was to quantitate the co-expression of two DNA repair proteins poly-ADP-ribose polymerase 1 and 2 (Parp-1 and Parp-2) by enzyme- linked immuno-sorbent assay (ELISA) in 60 endometrioid endometrial tumor samples and compare their expression to matched non-malignant endometrial tissue from the same corresponding donors from central Appalachia. Results We found that Parp-1 was significantly overexpressed in endometrial carcinoma relative to corresponding normal tissue. This overexpression implicates Parp inhibition therapy as a possible treatment for the disease. Our results also found a protective effect of native Parp-2 expression in non-malignant endometrial tissue with each 1 ng/mL increase in PARP-2 concentration in normal tissue was associated with a 10 % reduction in the hazard of tumor progression (HR = 0.90; p = 0.039) and a 21 % reduction in the hazard of death (HR = 0.79; p = 0.044). Conclusions This study demonstrated the over-expression of the druggable target Parp-1 in endometrial adenocarcinoma and observed a strong negative correlation of native Parp-2 expression and disease progression via the quantification of the Parp proteins using enzyme- linked immuno-sorbent assay (ELISA) assays.

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates&reg, Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix, Acellular bone composite discs, Allowash&reg, cancellous bone cubes, PLGA (poly lactic-co-glycolic acid), and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

A 38-year-old woman was found to have a large placental chorioangioma. The fetus was studied using ultrasound. The pregnancy became complicated by hydrops fetalis, polyhydramnios, and abruptio placenta. The infant delivered at 29 weeks' gestational age. The neonatal course was complicated by nonimmune hydrops fetalis, respiratory distress syndrome, anemia, pulmonary hemorrhage, intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia. The infant was discharged home with breastfeeding and off oxygen at 50 days of life. This case represents the multidisciplinary approach to the pregnancy complicated by a large placental chorioangioma and the resulting premature neonate with nonimmune hydrops fetalis.

High grade astrocytomas such as anaplastic astrocytoma and glioblastoma multiforme are aggressive central nervous system malignancies with a poor prognosis. Due to shortened survival times, their devastating effects are usually localized intracranially and rarely metastasize outside of the central nervous system. When metastases occur, they usually present in patients with longer survival times and they typically coincide with a primary site recurrence. We present a rare case of metastases from a high-grade astrocytoma/glioblastoma to the pleura, bones and liver within six months of diagnosis, without primary site recurrence.

Cancer progression is a complex multistep process comprising of angiogenesis of the primary tumor, its invasion into the surrounding stroma and its migration to distant organs to produce metastases. Nutritional compounds of the “capsaicinoid” family regulate angiogenesis, invasion and metastasis of tumors. Capsaicinoids display robust anti-angiogenic activity in both cell culture and mice models. However, conflicting reports exist about the effect of capsaicinoids on invasion of metastasis of cancers. While some published reports have described an anti-invasive and anti-metastatic role for capsaicinoids, others have argued that capsaicinoids stimulate invasion and metastasis of cancers. The present review article summarizes these findings involving the bioactivity of capsaicin in angiogenesis, invasion and metastasis of cancer. A survey of literature indicate that they are several articles summarizing the growth-inhibitory activity of capsaicinoids but few describe its effects on angiogenesis, invasion and metastasis in detail. Our review article fills this gap of knowledge. The discovery of a second generation of natural and synthetic capsaicin analogs (with anti-tumor activity) will pave the way to improved strategies for the treatment of several human cancers.

Eosinophilic esophagitis (EoE) has been recognized as a chronic disease of the esophagus with significant involvement of the mucosal immune system. Similar conditions in other gastrointestinal diseases have led to a malignant transformation (ie, inflammatory bowel disease, celiac disease, etc). MIB-1 (Ki-67) and p53 are monoclonal antibodies that are used to detect early markers for dysplastic changes, possibly leading to cancer development in adult patients with chronic gastroesophageal reflux disease (GERD), Barrett's esophagus, and/or the adenocarcinoma sequence of the esophagus. Only limited studies of these cell markers have been published in children with EoE. The aim of this study is to examine p53 and Ki-67 cell markers in children with EoE before and after medical therapy. The immunohistochemical staining of cell markers p53 and Ki-67 was examined in esophageal biopsies of children diagnosed with EoE, GERD, and normal esophagus. In addition, biopsies from adults with EoE and adenocarcinoma were used as positive controls. In 10 children who were successfully treated for EoE, immunohistochemical staining was compared before and after medical therapy. The immunohistochemical staining of p53 and Ki-67 was increased in children with EoE compared with children with a normal esophagus but not in children with GERD. Children with EoE posttherapy had significantly lower immunohistochemical staining for both markers compared to pretreatment staining. p53 and Ki-67 markers are associated with cell proliferation in children with EoE but do not represent a premalignant (dysplastic) condition of the esophagus.

Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin-based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti-cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data fosters hope for novel nutrition based combination therapies in SCLC.

Oncocytic neoplasms are tumors composed predominantly or exclusively of oncocytes (large polygonal cells with granular eosinophilic cytoplasm due to abnormal mitochondrial accumulation). These tumors are frequently reported in the thyroid, kidneys, and salivary glands. However, they are distinctly rare in the adrenal cortex. Oncocytic adrenocortical neoplasms (OAN) are classified regarding their biological behavior by their histological features according to the Lin-Weiss-Bisceglia system (LWB). Here, we report a case of OAN of borderline or uncertain malignant potential (BMP) with subsequently identified papillary thyroid carcinoma (PTC). A 34-year-old female with a nine-month history of fatigue presented with chest pain. A right adrenal mass was incidentally found while ruling out pulmonary embolism. A CT-guided adrenal biopsy, although not routinely indicated, was performed and interpreted as malignant with no definitive origin. Hormonal workup was unremarkable. PET-scan showed hypermetabolic adrenal mass with peak standardized uptake value of 15, suspicious of malignancy. A hypermetabolic thyroid nodule was also identified, but there was no evidence of metastatic disease. The patient underwent adrenalectomy, and the initial pathology report was interpreted as atypical pink cell tumor. A second pathology report from another laboratory favored OAN based on the morphology and immunohistochemical staining. While the histologic criteria of malignancy were not met, the large tumor size makes it compatible with BMP according to LWB criteria. A follow-up thyroid ultrasound revealed a complex thyroid nodule. A total thyroidectomy was performed, and pathology was consistent with PTC. Of interest, PTC frequently shows an increase in mitochondrial content, which is characteristic of oncocytic tumors. This case illustrates that OAN, although rare, should be considered in the differential diagnosis of adrenal masses. When OAN is identified, it should be classified regarding its biological behavior as benign or malignant using the LWB system and, eventually, the reticulin algorithm of Duregon, et al. Oncocytoma can be confirmed ultrastructurally or by immunohistochemistry. Studying the gene mutations in patients presenting with oncocytic malignancies and other tumors that demonstrate mitochondrial proliferation as PTC might help to understand the role of mitochondrial proliferation in cancer development.

2057Background: Over the past 20 years even with the aggressive standard of care (SoC) Stupp treatment protocol the prognosis of glioblastoma (GBM) has only minimally improved from 12 to 14 months....

Follicular dendritic cell tumor (FDCT) is an uncommon neoplasm that typically presents as a slow-growing, painless mass without systemic symptoms. Histologically, FDCT is characterized as a proliferation of spindle to ovoid cells having plump eosinophilic cytoplasm with indistinct borders and nuclei with vesicular or granular chromatin and small distinct nucleoli. The immunohistochemical profile of FDCT includes positive staining for CD21, CD23, CD35, vimentin, fascin, HLA-DR, epithelial membrane antigen, clusterin, and D2-40. Follicular dendritic cell tumor occurs primarily in lymphoid tissue; however, involvement of extranodal sites such as the tonsils, spleen, and gastrointestinal tract has been reported. Lung involvement typically represents metastatic disease with, to our knowledge, only 2 prior cases of extranodal primary FDCT of the lung reported. We report the third case of primary pulmonary FDCT arising in a 64-year-old woman.

Histologic criteria have a limited role in determining whether the synchronous bilateral breast carcinomas represent two primaries or a metastasis to the contralateral breast. We studied the molecular analysis of synchronous bilateral breast carcinoma and whether they are originating from a single or different clone. We examined 17 patients with breast carcinoma, including 12 patients with synchronous bilateral carcinomas and control group of 5 infiltrating ductal carcinomas with regional lymph node metastases. Mutations were quantitatively determined to detect loss of heterozygosity (LOH) and microsatellite size alterations for a broad panel of 15 markers, involving 10 chromosomes using polymerase chain reaction. The carcinomas were classified as de novo or metastasis based on three levels of concordance: (1) marker-affected tumors were considered concordant if 50% or more of the same markers were mutated, (2) same gene copy affected, and (3) temporal sequence of mutation acquisition. In synchronous bilateral breast carcinoma patients, molecular analysis showed discordant mutations in all cases, supporting the diagnosis of de novo bilateral primary breast carcinomas. In patients with lymph node metastases, the primary breast carcinoma and metastases shared the same mutations, revealing a metastatic lesion. In conclusion, the application of molecular technology may play an important role for the differential diagnosis of dual primary carcinomas vs a metastatic breast cancer to contralateral breast. In this study, synchronous bilateral breast cancers represent two independent primaries rather than metastatic events.

Lymph node metastases is an important prognostic indicator for disease progression and crucial for therapeutic strategies in the work-up of colorectal carcinoma. In this study, we investigated tumor lymphangiogenesis and vascular endothelial growth factor (VEGF) expression as predictive markers for the risk of lymph node metastasis and their relation to other prognostic parameters in colorectal carcinoma. Resected colorectal carcinomas from 90 patients were examined, including 30 patients without lymph node metastases, 30 with only lymph node metastases, and 30 with liver metastases. Cases were immunostained for CD31, D2-40, and VEGF. Positivity stained microvessels were counted in densely vascular/lymphatic foci (hot spots) at x 400 field (=0.17 mm2). Intensity of staining for VEGF was scored on a two-tiered scale. D2-40 lymphatic microvessel density demonstrated significant correlation with CD31 counts (20+/-9 vs 18+/-6/0.17 mm2 field, P

// Francesco Morra 1,2,* , Chiara Luise 1,* , Francesco Merolla 1,3 , Ina Poser 4 , Roberta Visconti 1 , Gennaro Ilardi 3 , Simona Paladino 2 , Hiroyuki Inuzuka 5 , Gianluca Guggino 6 , Roberto Monaco 7 , David Colecchia 8 , Guglielmo Monaco 6 , Aniello Cerrato 1 , Mario Chiariello 8 , Krista Denning 9 , Pier Paolo Claudio 10 , Stefania Staibano 3 and Angela Celetti 1 1 Istituto per l’Endocrinologia e l’Oncologia Sperimentale “Gaetano Salvatore”, CNR, Napoli, Italy 2 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita Federico II, Napoli, Italy 3 Dipartimento di Scienze Biomediche Avanzate, Universita Federico II, Napoli, Italy 4 Max Plank Institute, MPI-CBG Dresden, Germany 5 Harvard Medical School, Beth Israel Deaconess Medical Center, MA, USA 6 UOC Chirurgia Toracica, Azienda Ospedaliera di Rilievo Nazionale “A.Cardarelli”, Napoli, Italy 7 UOC Anatomia Patologica, Azienda Ospedaliera di Rilievo Nazionale “A.Cardarelli”, Napoli, Italy 8 Istituto Toscano Tumori, Core Research Laboratory, Siena, Italy 9 Department of Pathology, Joan C. Edwards Cancer Center, Huntington, WV, USA 10 Department of Biochemistry and Microbiology & Dept. of Surgery, Marshall University, Joan C. Edwards Cancer Center, Huntington, WV, USA * These authors have contributed equally to this work Correspondence to: Angela Celetti, email: // Keywords : CCDC6, FBXW7, USP7, mitotic kinases, cisplatinum Received : January 20, 2015 Accepted : March 03, 2015 Published : March 30, 2015 Abstract CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet. We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response. Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem-like cells (CSLCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy sensitivity assay (ChemoID), which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents. Two patients, a 21-year old male (patient 1) and a 5-month female (patient 2), affected by anaplastic WHO grade-III ependymoma were screened using the ChemoID assay. Patient 1 was found sensitive to the combination of irinotecan and bevacizumab, which resulted in a prolonged disease progression free period of 18 months. Following recurrence, the combination of various chemotherapy drugs was tested again with the ChemoID assay. We found that benzyl isothiocyanate (BITC) greatly increased the chemosensitivity of the ependymoma cells to the combination of irinotecan and bevacizumab. After patient 1 was treated for two months with irinotecan, bevacizumab and supplements of cruciferous vegetable extracts containing BITC, we observed over 50% tumoral regression in comparison with pre-ChemoID scan as evidenced by MRI. Patient 2 was found resistant to all treatments tested and following 6 cycles of vincristine, carboplatin, cyclophosphamide, etoposide, and cisplatin in various combinations, the tumor of this patient rapidly progressed and proton beam therapy was recommended. As expected animal studies conducted with patient derived xenografts treated with ChemoID screened drugs recapitulated the clinical observation. This assay demonstrates that patients with the same histological stage and grade of cancer may vary considerably in their clinical response, suggesting that ChemoID testing which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents could lead to more effective and personalized anticancer treatments in the future.

Objective To review the challenges encountered in the diagnostic work-up and management of patients with subclinical Cushing's syndrome (SCS) and bilateral adrenal masses to aid in the case description of a patient with SCS and adrenocorticotropic hormone (ACTH)-independent macronodular adrenal hyperplasia (AIMAH). Methods We describe our experience managing a patient with AIMAH and SCS. This case report is followed by an extensive review of the literature regarding differential diagnoses, work-up including adrenal venous sampling (AVS), and treatment of SCS with bilateral adrenal masses. Results A 51-year-old female who was diagnosed with recent onset hypertension and diabetes mellitus type 2 was evaluated for adrenal masses discovered incidentally on computed tomography (CT). She did not have any Cushingoid features. Magnetic resonance imaging (MRI) of abdomen was performed for further evaluation. Hormonal evaluation came back consistent with SCS. The AVS results were consistent with bilateral autonomous cortisol hypersecretion without lateralization. Collectively, the findings favored the diagnosis of bilateral AIMAH. A left adrenalectomy was performed, and the patient's clinical response was favorable with improvement in blood pressure (BP) accompanied by significant weight loss. Follow-up hormonal testing for autonomous cortisol hypersecretion was within the target range. Conclusions AIMAH is a rare cause of SCS. AVS is a useful diagnostic tool that helps localize the source of autonomous cortisol hypersecretion in ACTH-independent SCS with bilateral adrenal masses, especially if radiological features are inconclusive. Patients undergoing unilateral adrenalectomy should be followed for monitoring of clinical response, as well as progression of AIMAH in the contralateral adrenal gland. (Endocr Pract. 2013;19:515-521)

Acinic cell carcinoma (ACC) is an uncommon salivary gland neoplasm that generally displays an indolent growth pattern. Most cases arise in the major glands, particularly the parotid. However, it can arise from minor salivary glands in the oral cavity and aero-digestive tract. Although ACC is generally a low-grade malignant tumor, poorly differentiated and high-grade transformed variants exhibit a propensity for late recurrence and metastasis. There are no adequate clinical trials that define the optimal approach to patients with metastatic salivary gland tumors due to its rarity. Systemic therapy is reserved for cases where local therapy, such as radiation or metastasectomy, is not appropriate. Nevertheless, there is insufficient data in the literature regarding the chemotherapy of choice for metastatic ACC. In this article, we report a case of metastatic ACC of the right parotid gland that progressed on carboplatin and paclitaxel after partial response followed by doxorubicin and is currently on checkpoint inhibitor treatment.

e13028 Background: Glioblastoma multiforme (GBMs) are brain tumors with low survival rates due to the presence of chemoresistant Glioma Cancer Stem-Like Cells (GCSLCs). Development of a test that i...

e13012 Background: Cancer stem-like cells (CSLCs) in primary brain tumors can resist certain chemotherapies, thereby causing relapse of the disease. Thus, development of a test that identifies the ...

Eosinophilic esophagitis (EoE) has been recognized as a chronic disease of the esophagus in children and adults. Chronic inflammation of the esophagus may lead to malignant transformation of cells. Malignant or pre-malignant (dysplasia) conditions of the esophagus in EoE has not been reported. p53, a mutated intracellular oncogene marker, and Ki-67, a nuclear antigen associated with cell proliferation, were found to be increased in patients with Barrett's esophagus and esophageal cancer. Aim: To evaluate the presence of p53 and Ki-67 in children with EoE. Materials: Esophageal biopsies of children with EoE (Test Group), children with GERD (Control 1), adults with EoE (Control 2), adults with esophageal cancer (Control 3), and children with normal esophagus (Control 4), comprised our patient population. The immunreactivity of monoclonal oncogene p53 (DAKO, DO-7) and monoclonal Ki-67 cell marker (DAKO, Clone MIB-1) were utilized. Immunoreactivity of p53 was considered positive when at least 10% of the cells stained positive (Wang 1993). Ki-67 immunoreactivity was calculated as a percentage of positive cells per at least 500 cells (Feith 2004). Results: A total of 50 esophageal biopsies were available, 25 children with EoE, 15 children with GERD, 15 children with normal esophagus, 3 adults with EoE, and 2 adults with esophageal cancer. p53 was negative in normal mucosa but was Ki-67 positive in the basal layer (proliferative zone). Adult biopsies with esophageal cancer were positive for both markers. Immunoreactivity of p53 and Ki-67 was significantly higher in EoE vs. normal esophagus (Table). Conclusion: Oncogene p53 and Ki-67 immunoreactivity were higher in the EoE compared to the GERD and the normal group. Our data suggested an active cell proliferation in the esophagus of children with EoE and GERD. Whether the positive immunoreactivity of p53 in EoE children represents a real genetic mutation or false positive results is yet to be resolved (Rice 1994). Oncogene immunoreactivity in EoE