Lyska Emerson

Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia

Efforts to model pancreatic cancer in mice have focused on mimicking genetic changes found in the human disease, particularly the activating KRAS mutations that occur in pancreatic tumors and their putative precursors, pancreatic intraepithelial neoplasia (PanIN). Although activated mouse Kras mutations induce PanIN lesions similar to those of human, only a small minority of cells that express mutant Kras go on to form PanINs. The basis for this selective response is unknown, and it is similarly unknown what cell types in the mature pancreas actually contribute to PanINs. One clue comes from the fact that PanINs, unlike most cells in the adult pancreas, exhibit active Notch signaling. We hypothesize that Notch, which inhibits differentiation in the embryonic pancreas, contributes to PanIN formation by abrogating the normal differentiation program of tumor-initiating cells. Through conditional expression in the mouse pancreas, we find dramatic synergy between activated Notch and Kras in inducing PanIN formation. Furthermore, we find that Kras activation in mature acinar cells induces PanIN lesions identical to those seen upon ubiquitous Kras activation, and that Notch promotes both initiation and dysplastic progression of these acinar-derived PanINs, albeit short of invasive adenocarcinoma. At the cellular level, Notch/Kras coactivation promotes rapid reprogramming of acinar cells to a duct-like phenotype, providing an explanation for how a characteristically ductal tumor can arise from nonductal acinar cells.

Contrast-Enhanced MRI-Guided Photodynamic Cancer Therapy with a Pegylated Bifunctional Polymer Conjugate

PurposeTo study contrast-enhanced MRI guided photodynamic therapy with a pegylated bifunctional polymer conjugate containing an MRI contrast agent and a photosensitizer for minimally invasive image-guided cancer treatment.MethodsPegylated and non-pegylated poly-(l-glutamic acid) conjugates containing mesochlorin e6, a photosensitizer, and Gd(III)-DO3A, an MRI contrast agent, were synthesized. The effect of pegylation on the biodistribution and tumor targeting was non-invasively visualized in mice bearing MDA-MB-231 tumor xenografts with MRI. MRI-guided photodynamic therapy was carried out in the tumor bearing mice. Tumor response to photodynamic therapy was evaluated by dynamic contrast enhanced MRI and histological analysis.ResultsThe pegylated conjugate had longer blood circulation, lower liver uptake and higher tumor accumulation than the non-pegylated conjugate as shown by MRI. Site-directed laser irradiation of tumors resulted in higher therapeutic efficacy for the pegylated conjugate than the non-pegylated conjugate. Moreover, animals treated with photodynamic therapy showed reduced vascular permeability on DCE-MRI and decreased microvessel density in histological analysis.ConclusionsPegylation of the polymer bifunctional conjugates reduced non-specific liver uptake and increased tumor uptake, resulting in significant tumor contrast enhancement and high therapeutic efficacy. The pegylated poly(l-glutamic acid) bifunctional conjugate is promising for contrast enhanced MRI guided photodynamic therapy in cancer treatment.

Serum Osteopontin and Tissue Inhibitor of Metalloproteinase 1 as Diagnostic and Prognostic Biomarkers for Pancreatic Adenocarcinoma

Objectives Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 5%. There is an urgent need for early detection while the tumors are small and surgically resectable. We assessed serum osteopontin (OPN) and tissue inhibitor of metalloproteinase 1 (TIMP-1) as possible diagnostic and prognostic biomarkers in a novel cohort of patients with pancreatic cancer. Methods Osteopontin and TIMP-1 levels were determined in sera from 86 patients with PDAC, 86 healthy control subjects, and 48 patients with chronic pancreatitis. Regression models were used to relate OPN and TIMP-1 to sex, age, stage, class, and treatment. Survival analyses were performed using univariate and multivariate Cox models. Results The serum levels of both OPN and TIMP-1 distinguished PDAC from chronic pancreatitis (P ⩽ 0.0001) and healthy control subjects (P < 0.0001). The serum levels of both OPN and TIMP-1 also distinguished early-stage resectable PDAC cases from chronic pancreatitis (P < 0.04) and healthy control subjects (P < 0.01). High serum levels of OPN were significantly correlated with reduced patient survival. Conclusions Serum OPN and TIMP-1 have use as diagnostic biomarkers in PDAC. Our data suggest a potential benefit of using OPN, TIMP-1, and CA 19-9 in a panel to improve diagnostic accuracy in PDAC.

Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration

Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.

A Comparison of Immunohistochemical Stain Quality in Conventional and Rapid Microwave Processed Tissues

Same-day turnaround of pathology specimens is desirable in this era of managed care, and rapid microwave tissue processing produces histologic features of a quality equivalent to overnight processing. We studied whether microwave-assisted rapid tissue processing adversely affects the quality of immunohistochemical staining. We selected 30 specimens (20 neoplastic and 10 nonneoplastic) from our routine surgical pathology workload. Paired large tissue blocks were made from each specimen type, one for microwave-assisted rapid processing and one for conventional processing. Two microarrays of 60 punches each were made from the donor blocks. The microarray blocks were examined for intensity and extent of staining by 44 commonly used antibodies. Slides were reviewed independently by 2 pathologists blinded to the type of processing used. In 5,280 tissue punches examined, we found a high degree of concordance in quality, as measured by intensity and extent of immunohistochemical staining, between microwave and routinely processed tissues. Our study demonstrates that quality of immunohistochemical staining is similar between rapid microwave and conventional processing. The potential need for immunohistochemical analysis is not a contraindication for microwave-assisted rapid tissue processing.

Collecting duct carcinoma arising in association with bk nephropathy post-transplantation in a pediatric patient. a case report with immunohistochemical and in situ hybridization study

Abstract:  The development of malignancy in a renal transplant graft is an uncommon phenomenon. A renal neoplasm developing in the adult donor kidney of a pediatric transplant recipient has only rarely been reported. We report a case of collecting duct carcinoma arising in association with BK virus nephropathy in an adult living‐related donor renal allograft to a pediatric recipient. Our case is the second report of neoplasia occurring in association with BK virus nephropathy post‐transplantation, suggesting that BK virus may play a role in oncogenesis. It has been proposed that the T‐Ag protein encoded by the polyomavirus family of viruses disrupts chromosomal integrity, creating oncogenes, and inactivating tumor suppressor genes. In our study, immunohistochemical staining with antibody directed against BK virus large T antigen showed nuclear staining within urothelium, tubular epithelium, tubular intraepithelial neoplasia, and invasive carcinoma. In situ hybridization did not identify BK virus DNA within neoplastic cells. T‐Ag protein expression has been shown to be tumor‐specific in bladder, gastric, and colorectal cancers. The finding of T‐Ag protein expression in both intraepithelial and invasive neoplastic tissues in our case raises the possibility of BK virus as a causative agent in oncogenesis.

Characterization of tumor angiogenesis with dynamic contrast‐enhanced MRI and biodegradable macromolecular contrast agents in mice

The efficacy of polydisulfide‐based biodegradable macromolecular contrast agents for characterizing tumor angiogenesis was investigated in a mouse model using dynamic contrast‐enhanced MRI (DCE‐MRI). Biodegradable macromolecular MRI contrast agents, gadopentetate dimeglumine (Gd‐DTPA) cystamine copolymers (GDCC), and Gd‐DTPA cystine copolymers (GDCP), with molecular weights of 20 and 70 kDa were used in the study. Gadodiamide (Gd [DTPA‐BMA]) and albumin labeled with Gd‐DTPA [albumin‐(Gd‐DTPA)] were used as the controls. The DCE‐MRI studies were performed in nude mice bearing prostate tumor xenografts from the MDA‐PCa‐2b cell line. Tumor angiogenic kinetic parameters, including endothelial transfer coefficient (KPS), fractional tumor plasma volume (fPV), and permeability surface area product (PS), were estimated from the DCE‐MRI data using a two‐compartment model. The KPS and fPV values estimated by the biodegradable macromolecular contrast agents were between those estimated by Gd(DTPA‐BMA) and albumin‐(Gd‐DTPA). The parameters estimated by the agent with a slow degradation rate and high molecular weight, GDCP‐70 (KPS = 2.09 ± 0.50 ml/min/100 cc and fPV = 0.075 ± 0.021), were closer to those by albumin‐(Gd‐DTPA) (KPS = 1.43 ± 0.64 ml/min/100 cc and fPV = 0.044 ± 0.007) than by other agents with relatively low molecular weight or rapid degradation rate. The polydisulfide‐based biodegradable macromolecular contrast agents are promising for characterizing tumor vascularity and angiogenesis with DCE‐MRI. Magn Reson Med 60:1347–1352, 2008.

Neutralization of cardiac toxins oleandrin, oleandrigenin, bufalin, and cinobufotalin by digibind: Monitoring the effect by measuring free digitoxin concentrations

Oleandrin plant poisoning is common in children and the plant extract is used in Chinese medicines. The toxicity is due to oleandrin and the deglycosylated metabolite oleandrigenin. Bufalin and cinobufotalin (toad cardiac toxins) are also widely used in Chinese medicines like Chan SU, and Lu-Shen -WU. Severe toxicity from bufalin after consumption of toad soup has been reported. Taking advantage of structural similarities of these toxins with digitoxin, we demonstrated that these compounds can be rapidly detected in blood by the fluorescence polarization immunoassay for digitoxin. The cross reactivities of these compounds with digoxin assay were much lower. For example, when a drug free serum was supplemented with 10 microg/ml of oleandrin, we observed 127.7 ng/ml of digitoxin equivalent but only 2.4 ng/ml of digoxin equivalent concentration. Digibind neutralized all cardiac toxins studied as evidenced by significant fall of free concentrations. When aliquots of serum pool containing 50.0 microg/ml of oleandrin were supplemented with 0, 10.0, 25.0, 50.0, 100, and 200 microg/ml of digibind, the mean free concentrations were 30.6, 23.3, 16.0, 10.7, 7.8 and 5.5 microg/ml respectively. Similarly, with 50.0 microg/ml of oleandrigenin (total concentration: 36.2 ng/ml), the free concentration was 14.5 ng/ml digitoxin equivalent in the absence of digibind and 5.4 ng/ml in the presence of 200 microg/ml of digibind. In another specimen containing 500 ng/ml bufalin (total concentration: 156.9 ng/ml), the free concentration was 8.6 ng/ml in the absence of digibind and none detected in the presence of 100.0 microg/ml digibind. Because such neutralization may also occur in vivo, digibind may be useful in treating patients exposed to these toxins.

KIT mutations in ocular melanoma: Frequency and anatomic distribution

KIT mutations are known to occur in ∼15% of chronic sun damaged cutaneous, mucosal, and acral melanomas. Melanomas with demonstrated activating mutations in KIT or platelet-derived growth factor receptor A (PDGFRA) may benefit from treatment with tyrosine kinase inhibitors. Currently, the limited data regarding KIT mutational status in ocular melanoma suggest that activating mutations are extremely rare. PDGFRA mutational status in ocular melanoma has not been determined. Seventy-five ocular melanomas (53 choroidal, 6 iris, 11 ciliary body, and 5 conjuctival) were selected from the files of the Department of Ophthalmology. High-resolution melting curve analysis and sequencing were performed to detect mutations in KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18. Results of mutational analysis were correlated with anatomical site and KIT (CD117) immunohistochemistry. Eight of 75 (11%) ocular melanomas contained mutations in either the KIT or PDGFRA gene. Five of 53 (9%) choroidal melanomas were associated with mutations (KIT exon 11=3; KIT exon 17=1; PDGFRA intron 18=1). Two of six (33%) iris melanomas and a single (9%) ciliary body melanoma harbored KIT exon 11 mutations. No mutations were identified in conjunctival melanomas. The distribution of KIT and PDGFRA mutations by ocular melanoma anatomical site did not reach statistical significance (P=0.393) CD117 positivity was not predictive of KIT mutational status as only 6 of 58 (10%) CD177-positive tumors harbored KIT mutations. In addition, a KIT exon 17 mutation was identified in one CD117-negative tumor. KIT and PDGFRA mutations do occur in ocular melanomas at a frequency (11%) that is similar to acral and mucosal melanomas. Limited correlation of CD117 positivity with mutational status suggests that all ocular melanomas should undergo mutational analysis to determine if imatinib therapy is appropriate.

Serum Platelet Factor 4 is an Independent Predictor of Survival and Venous Thromboembolism in Patients with Pancreatic Adenocarcinoma

Background: Improved diagnostic, predictive, and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC) are urgently needed. Platelet factor 4 (PF4) has been proposed as a diagnostic biomarker for PDAC. We assessed the diagnostic and prognostic potential of serum PF4 levels in PDAC patients. Methods: Serum PF4 levels were determined by enzyme-linked immunosorbent assay in an initial cohort of 62 PDAC patients, 62 healthy control subjects, and 34 chronic pancreatitis patients. A second validation set consisted of 71 PDAC patients. Linear regression models were used to relate PF4 to class, gender, age, stage, platelet count, and diagnosis. Survival analyses were done using univariate and multivariate Cox models. Results: In the initial cohort, serum PF4 levels distinguished PDAC from chronic pancreatitis patients (P = 0.011), but not from healthy control subjects (P = 0.624). In PDAC patients, high serum PF4 level significantly predicted decreased survival independent of all covariates examined (P < 0.01). The prognostic relationship of serum PF4 levels remained significant in the validation set. Venous thromboembolism (VTE) occurred in 20% of the 133 PDAC patients. The VTE risk was higher in subjects with elevated PF4 levels (P = 0.009). Conclusions: Serum PF4 is shown for the first time to be prognostic for survival in PDAC patients. High PF4 is associated with an increased risk for the development of VTE. Impact: Serum PF4 levels may be useful for patient stratification and for directing treatment options in patients with pancreatic cancer including anticoagulation prophylaxis. The relationship between high PF4 levels and poorer outcomes requires further study. Cancer Epidemiol Biomarkers Prev; 19(10); 2605–10. ©2010 AACR.