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1.  Detergent-free Decellularized Nerve Grafts for Long-gap Peripheral Nerve Reconstruction 
Long-gap peripheral nerve defects arising from tumor, trauma, or birth-related injuries requiring nerve reconstruction are currently treated using nerve autografts and nerve allografts. Autografts are associated with limited supply and donor-site morbidity. Allografts require administration of transient immunosuppressants, which has substantial associated risks. To overcome these limitations, we investigated the use of detergent-free decellularized nerve grafts to reconstruct long-gap nerve defects in a rodent model and compared it with existing detergent processing techniques.
Nerve grafts were harvested from the sciatic nerves of 9 donor rats. Twenty-four recipient rats were divided into 4 groups (6 animals per group): (1) nerve grafts (NG, positive control), (2) detergent-free decellularized (DFD) grafts, (3) detergent decellularized grafts, and (4) silicone tube conduits (negative control). Each recipient rat had a 3.5-cm graft or conduit sutured across a sciatic nerve transection injury. All animals were harvested at 12 weeks postimplantation for functional muscle analysis and nerve histomorphometry.
Histomorphometry results indicated maximum growth in NG when compared with other groups. DFD and detergent decellularized groups showed comparable regeneration at 12 weeks. Silicone tube group showed no regeneration as expected. Muscle force data indicated functional recovery in NG and DFD groups only.
This study describes a detergent-free nerve decellularization technique for reconstruction of long-gap nerve injuries. We compared DFD grafts with an established detergent processing technique and found that DFD nerve grafts are successful in promoting regeneration across long-gap peripheral nerve defects as an alternative to existing strategies.
PMCID: PMC4236362  PMID: 25426384
2.  Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold 
ACS Medicinal Chemistry Letters  2013;4(5):491-496.
Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp2 hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>103) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.
PMCID: PMC4027141  PMID: 24900696
Fibroblast activation protein (FAP); dipeptidyl peptidase IV (DPPIV); prolyl oligopeptidase (PREP); seprase
3.  An Activatable Near Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha 
Bioconjugate chemistry  2012;23(8):1704-1711.
Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.
PMCID: PMC3419799  PMID: 22812530
FAPα; NIRF; glioma; activatable probe; optical imaging
4.  Symptomatic cardiac toxicity is predicted by dosimetric and patient factors rather than changes in 18F-FDG PET determination of myocardial activity after chemoradiotherapy for esophageal cancer 
Radiotherapy and Oncology  2012;104(1):72-77.
To determine factors associated with symptomatic cardiac toxicity in patients with esophageal cancer treated with chemoradiotherapy.
Material and Methods
We retrospectively evaluated 102 patients treated with chemoradiotherapy for locally advanced esophageal cancer. Our primary endpoint was symptomatic cardiac toxicity. Radiation dosimetry, patient demographic factors, and myocardial changes seen on 18F-FDG PET were correlated with subsequent cardiac toxicity. Cardiac toxicity measured by RTOG and CTCAE v3.0 criteria was identified by chart review.
During the follow up period, 12 patients were identified with treatment related cardiac toxicity, 6 of which were symptomatic. The mean heart V20 (79.7% vs. 67.2%, p=0.05), V30 (75.8% vs. 61.9%, p=0.04), and V40 (69.2% vs. 53.8%, p=0.03) were significantly higher in patients with symptomatic cardiac toxicity than those without. We found the threshold for symptomatic cardiac toxicity to be a V20, V30 and V40 above 70%, 65% and 60%, respectively. There was no correlation between change myocardial SUV on PET and cardiac toxicity, however, a greater proportion of women suffered symptomatic cardiac toxicity compared to men (p=0.005).
A correlation did not exist between percent change in myocardial SUV and cardiac toxicity. Patients with symptomatic cardiac toxicity received significantly greater mean V20, 30 and 40 values to the heart compared to asymptomatic patients. These data need validation in a larger independent data set.
PMCID: PMC3389132  PMID: 22682539
Chemoradiotherapy; Esophageal cancer; Cardiac Toxicity
5.  Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors1 
Translational Oncology  2012;5(6):422-429.
Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.
PMCID: PMC3542838  PMID: 23323157
6.  Evolution of Tumor Invasiveness: The Adaptive Tumor Microenvironment Landscape Model 
Cancer research  2011;71(20):6327-6337.
Interactions between cancer cells and their microenvironment are crucial for promoting tumor growth and invasiveness. In the tumor adaptive landscape model, hypoxic and acidic microenvironmental conditions reduce the fitness of cancer cells and significantly restrict their proliferation. This selects for enhanced motility as cancer cells may evolve an invasive phenotype if the consequent cell movement is rewarded by proliferation. Here, we used an integrative approach combining a mathematical tumor adaptive landscape model with experimental studies to examine the evolutionary dynamics that promote an invasive cancer phenotype. Computer simulation results hypothesized an explicit coupling of motility and proliferation in cancer cells. The mathematical modeling results were also experimentally examined by selecting Panc-1 cells with enhanced motility on a fibroblast-derived 3D matrix for cells that move away from the unfavorable metabolic constraints. After multiple rounds of selection, the cells that adapted through increased motility were characterized for their phenotypic properties compared to stationary cells. Microarray and gene depletion studies demonstrated the role of Rho-GDI2 in regulating both cell movement and proliferation. Together, this work illustrates the partnership between evolutionary mathematical modeling and experimental validation as a potentially useful approach to study the complex dynamics of the tumor microenvironment.
PMCID: PMC3197231  PMID: 21859828
Adaptive landscape model; tumor microenvironment; cell motility; 3D matrix; Rho-GDI2
7.  Quantitative MRI using T1ρ and T2 in human osteoarthritic cartilage specimens: Correlation with biochemical measurements and histology 
Magnetic resonance imaging  2010;29(3):324-334.
A direct correlation between T1ρ, T2 and quantified proteoglycan and collagen contents in human osteoarthritic cartilage has yet to be documented. We aimed to investigate the orientation effect on T1ρ and T2 values in human osteoarthritic cartilage; and to quantify the correlation between T1ρ, T2, versus biochemical composition and histology in human osteoarthritic cartilage.
Materials and Methods
Thirty-three cartilage specimens were collected from patients who underwent total knee arthroplasty due to severe osteoarthritis, and scanned with a 3T MR scanner for T1ρ and T2 quantification. Nine specimens were scanned at three different orientations with respect to the B0 : 0°, 90°, and 54.7°. Core punches were taken after MRI. Collagen and proteoglycan contents were quantified using biochemical assays. Histology sections were graded using Mankin scores. The correlation between imaging parameters, biochemical contents and histological scores were studied.
Both mean T1ρ and T2 at 54.7° were significantly higher than those measured at 90° and 0°, with T1ρ showing a less increase compared to T2. R1ρ (1/T1ρ) values had a significant, but moderate correlation with proteoglycan contents (R = 0.45, P = 0.002), while R2 (1/T2) was not correlated with proteoglycan. No significant correlation was found between relaxation times (T1ρ or T2) and collagen contents. The T1ρ values of specimen sections with high Mankin scores were significantly higher than those with low Mankin scores (P < 0.05)
Quantitative MRI has a great potential to provide non-invasive imaging biomarkers for cartilage degeneration in OA.
PMCID: PMC3058148  PMID: 21130590
Osteoarthritis; Magnetic Resonance Imaging; Cartilage matrix; T1rho; T2
8.  Phase II and Coagulation Cascade Biomarker Study of Bevacizumab with or without Docetaxel in Patients with Previously Treated Metastatic Pancreatic Adenocarcinoma 
Treatment options are limited for advanced pancreatic cancer progressive after gemcitabine therapy. The vascular endothelial growth factor (VEGF) pathway is biologically important in pancreatic cancer, and docetaxel has modest anti-tumor activity. We evaluated the role of the anti-VEGF antibody bevacizumab as second-line treatment for patients with metastatic pancreatic cancer.
Patients with metastatic adenocarcinoma of the pancreas who had progressive disease on a gemcitabine-containing regimen were randomized to receive bevacizumab alone or bevacizumab in combination with docetaxel.
Thirty-two patients were enrolled; 16 to bevacizumab alone (Arm A) and 16 to bevacizumab plus docetaxel (Arm B). Toxicities were greater in Arm B with the most common grade 3/4 nonhematologic toxicities including fatigue, diarrhea, dehydration and anorexia. No confirmed objective responses were observed. At 4 months, 2/16 patients in Arm A and 3/16 in Arm B were free from progression. The study was stopped according to the early stopping rule for futility. Median PFS and OS were 43 days and 165 days in Arm A and 48 days and 125 days in Arm B. Elevated D-dimer levels and thrombin-antithrombin complexes were associated with decreased survival and increased toxicity.
Bevacizumab with or without docetaxel does not have antitumor activity in gemcitabine-refractory metastatic pancreatic cancer. Baseline and on-treatment D-dimer and thrombin-antithrombin complex levels are associated with increased toxicity and decreased survival.
PMCID: PMC3030655  PMID: 20458210
9.  Evaluation of bone bruises and associated cartilage in ACL injured and reconstructed knees using quantitative T1ρ MRI: One-year cohort study 
To quantitate bone marrow edema-like lesions (BMEL) and the radiologic properties of cartilage in knees with acute anterior cruciate ligament (ACL) injuries using T1ρ MRI over a 1 year time period.
9 patients with ACL injuries were studied. MRI were acquired within 8 weeks of the injury, after which ACL reconstruction surgery was performed. Images were then acquired 0.5, 6, and 12 months following reconstructions. The volume and signal intensity of BMEL were quantified at baseline and follow up exams. T1ρ values were quantified in cartilage overlying the BMEL (OC) and compared to surrounding cartilage (SC) at all time-points.
BMEL were most commonly found in the lateral tibia and lateral femoral condyle. Nearly 50% of BMEL resolved over 1-year. The T1ρ values of the OC in the lateral tibia, medial tibia, and medial femoral condyle were elevated compared to respective regions in SC at all time points, significant only in the lateral tibia (P < 0.05). The opposite results were found in the lateral femoral condyle. For the medial tibia and medial femoral condyle, none of the time periods were significantly different. The percent increase in T1ρ values of OC in the lateral tibia was significantly correlated to BMEL-volume (r = 0.74, P < 0.05). At 1-year, the OC in the lateral tibia, medial tibia, and medial femoral condyle showed increased T1ρ values despite improvement of BMEL.
In patients following ACL tear and reconstruction, (1) the cartilage overlying BMEL in the lateral tibia experiences persistent T1ρ signal change immediately after acute injuries and at 1-year follow up despite BMEL improvement. (2) The superficial layers of the overlying cartilage demonstrate greater matrix damage than the deep layers, and (3) the volume of the BMEL may predict the severity of the overlying matrix's damage in the lateral tibia. T1ρ is capable of quantitatively and noninvasively monitoring this damage and detecting early cartilage changes in the lateral tibia over time.
Level of Evidence
PMCID: PMC3011041  PMID: 21035995
10.  FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells 
BMC Cancer  2011;11:245.
Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions.
We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer.
We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses.
We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated.
Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.
PMCID: PMC3141768  PMID: 21668992
11.  Decreased Posttreatment SUV on PET Scan Is Associated With Improved Local Control in Medically Inoperable Esophageal Cancer 
The relationship between local, regional, or distant disease control (LC, RC, DC) and maximal posttreatment standardized uptake value (SUVmax) in patients with esophageal cancer has not been elucidated. This study was initiated to explore whether a decrease in SUV on positron emission tomography-computed tomography (PET-CT) scan is associated with LC, RC, or DC in patients with esophageal carcinoma treated with definitive chemoradiotherapy.
Medical records of 40 patients with inoperable esophageal cancer treated with definitive intent and who underwent pre- and posttreatment PET-CT scans were reviewed. The histology, nodal status, tumor location, and radiotherapy (RT) dose were investigated as variables to determine a relationship between SUVmax and LC, RC, and DC as well as disease-free survival (DFS).
Decreased posttreatment SUVmax on PET scan (P = .02) and increased RT dose (P = .009) were the only significant predictors of improved LC on univariate analysis. Mean RT doses in patients with no evidence of disease or with local, regional, or distant recurrences were 5,244, 4,580, 5,094, and 4,968, respectively. Decreased posttreatment SUV (P = .03) and increased RT dose (P = .008) were also associated with an improvement in DFS. Furthermore, decreased posttreatment SUVmax correlated with an improvement in LC (hazard ratio [HR] = 1.3, 95% confidence interval [CI] = 1.03–1.6, P = .03) as well as DFS (HR = 1.3, 95% CI = 1.03–1.6, P = .03). These findings were maintained on multivariate analysis.
Posttreatment decrease in SUV is associated with LC and DFS in esophageal cancer patients receiving definitive chemoradiotherapy. RT dose was also associated with both LC and DFS. The prognostic significance of these findings warrants prospective confirmation.
PMCID: PMC3201642  PMID: 22043323
12.  A Phase I Dose-Escalation Study of Danusertib (PHA-739358) Administered as a 24-hour Infusion With and Without G-CSF in a 14-day Cycle in Patients with Advanced Solid Tumors 
This study was conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of the intravenous pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors.
Experimental Design
In Part 1, patients received escalating doses of danusertib (24-h infusion every 14 days) without filgrastim (G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was performed in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3).
Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose limiting toxicity in Part 1 without G-CSF. Most other adverse events were grade 1–2, occurring at doses ≥360 mg/m2 with similar incidence in parts 1 and 2. The MTD without G-CSF is 500 mg/m2. The recommended phase 2 dose (RP2D) in Part 2 with G-CSF is 750 mg/m2. Danusertib demonstrated dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18–26 hours. pH3 modulation in skin biopsies was observed at ≥500 mg/m2. One patient with refractory small cell lung cancer (1000 mg/m2 with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline.
Danusertib was well tolerated with target inhibition in skin at ≥500 mg/m2. Preliminary evidence of anti-tumor activity, including a PR and several occurrences of prolonged stable disease (SD), was seen across a variety of advanced refractory cancers. Phase II studies are ongoing.
PMCID: PMC2826106  PMID: 19825950
Danusertib; PHA-739358; Aurora Kinase Inhibitor; phase I trial; solid tumors
13.  Correlation of molecular response as measured by 18-FDG PET with outcome after chemo-radiation in patients with esophageal carcinoma 
Determine if 18-FDG PET-CT scans predict pathologic complete response, disease-free and overall survival in patients with esophageal carcinoma undergoing definitive or pre-operative chemoradiation.
Material & Methods
Patients with esophageal carcinoma presenting for definitive or pre-operative treatment undergoing pre- and post-treatment 18-FDG PET-CT scans were retrospectively reviewed. Histology, T-stage, nodal status, radiation dose, days from end of radiation to PET scan and surgery were the variables investigated to determine a relationship to baseline SUV of the primary tumor at the time of diagnosis. We also attempted to determine if a relationship existed between % decrease SUV and pathologic complete response, overall and disease-free survival.
Eighty-one patients, 14 female and 67 male, underwent 18-FDG PET-CT scanning prior to treatment and 63 had post-treatment scans. T-stage and tumor location predicted in univariate but not multivariate analysis for initial SUV. Sixty-six percent of patients with a post-chemoradiation SUV <2.5 had tumor seen in the surgical specimen and 64% of patients had positive lymph nodes at surgery not imaged on the post-chemoradiation PET scan. A trend existed for post-treatment SUV and days from radiation to surgery to predict for pathologic complete response, p=0.09 and p=0.08, respectively. Post-treatment SUV predicted for disease-free survival in the definitive chemoradiation group, p=0.01.
A correlation existed between depth of tumor invasion and baseline SUV level. Post-treatment SUV predicted for disease-free survival in the definite chemoradiation group. Caution should be exercised in utilizing post-treatment PET scans to determine the necessity of surgical resection.
PMCID: PMC2933372  PMID: 17532577
Molecular imaging; esophageal cancer; chemoradiation
14.  Identification and characterization of the promoter of fibroblast activation protein 
Fibroblast activation protein (FAP) is a type II integral membrane glycoprotein belonging to the serine protease family. It is selectively expressed by tumor stromal fibroblasts and transiently in the fibroblasts of healing wounds. FAP has been shown to modulate growth, differentiation, adhesion, and metastasis of tumor cells. Despite the importance of FAP in cancer, the mechanisms that govern its expression have not been defined. In this study, we determined the transcription start site of the FAP gene and identified a 2-kb segment with promoter activity in cells expressing FAP. Truncation of this fragment revealed that the core promoter activity resided in a 245-bp fragment surrounding the transcription start site. Electrophoretic mobility shift assay showed that EGR1 binds to the FAP promoter. Mutation of the EGR1 site within this fragment significantly decreased the promoter activity of FAP and eliminated EGR1 binding. Down-regulation of EGR1 resulted in a significant reduction in endogenous FAP mRNA expression. These findings identify the basal transcriptional requirements of FAP gene expression and show EGR1 is an important regulator of FAP expression.
PMCID: PMC2880831  PMID: 20515787
Fibroblast activation protein; Promoter
15.  Diagnostic performance of in vivo 3-T MRI for articular cartilage abnormalities in human osteoarthritic knees using histology as standard of reference 
European radiology  2008;18(10):2292-2302.
The purpose of this study was (1) to evaluate the sensitivity, specificity and accuracy of sagittal in vivo 3-T intermediate-weighted fast spin-echo (iwFSE) sequences in the assessment of knee cartilage pathologies using histology as the reference standard in patients undergoing total knee replacement, and (2) to correlate MR imaging findings typically associated with osteoarthritis such as bone marrow edema pattern (BMEP) and cartilage swelling with histological findings. Tibial plateaus and femoral condyles of eight knees of seven patients were resected during surgery, and sagittal histological sections were prepared for histology. Preoperative MRI findings were compared to the corresponding region in histological sections for thickness, surface integrity and signal pattern of cartilage, and histological findings in areas of BMEP and swelling were documented. The overall sensitivity, specificity and accuracy were 72%, 69% and 70% for thickness, 69%, 74% and 73% for surface and 36%, 62% and 45% for intracartilaginous signal pattern. For all cases of BMEP on MRI subchondral ingrowth of fibrovascular tissue and increased bone remodeling were observed. MRI using fat-saturated iwFSE sequences showed good performance in assessing cartilage thickness and surface lesions, while signal changes of cartilage were not suited to characterize the severity of cartilage degeneration as validated by histology.
PMCID: PMC2838768  PMID: 18491096
Cartilage; MRI; Comparative study; Histology
16.  Photodynamic Molecular Beacon Triggered by Fibroblast Activation Protein on Cancer-associated Fibroblasts for Diagnosis and Treatment of Epithelial Cancers 
Journal of medicinal chemistry  2009;52(2):358-368.
Fibroblast activation protein (FAP) is a cell-surface serine protease highly expressed on cancer-associated fibroblasts of human epithelial carcinomas but not on normal fibroblasts, normal tissues, and cancer cells. We report herein a novel FAP-triggered photodynamic molecular beacon (FAP-PPB) comprising of a fluorescent photosensitizer and a black hole quencher 3 linked by a peptide sequence (TSGPNQEQK) specific to FAP. FAP-PPB was effectively cleaved by both human FAP and murine FAP. Using the HEK293 transfected cells (HEK-mFAP, FAP+; HEK-Vector, FAP−), systematic in vitro and in vivo experiments validated the FAP-specific activation of FAP-PPB in cancer cells and mouse xenografts, respectively. FAP-PPB was cleaved by FAP, allowing fluorescence restoration in FAP-expressing cells, while leaving non-expressing FAP cells undetectable. Moreover, FAP-PPB showed FAP-specific photocytotoxicity toward HEK-mFAP cells whereas it was non-cytotoxic toward HEK-Vector cells. This study suggests that the FAP-PPB is a potentially useful tool for epithelial cancer detection and treatment.
PMCID: PMC2773291  PMID: 19093877
Fibroblast Activation Protein (FAP); Cancer-associated Fibroblast (CAF); Molecular Beacon; In Vivo Imaging; Photodynamic Therapy (PDT)
17.  A Phase I Study of the Safety and Pharmacokinetics of Trabectedin in Combination With Pegylated Liposomal Doxorubicin in Patients With Advanced Malignancies 
To determine the maximum tolerated dose (MTD), safety, potential pharmacokinetic (PK) interactions, and effect on liver histology of trabectedin in combination with pegylated liposomal doxorubicin (PLD) for advanced malignancies.
Patients and Methods
Entry criteria for the 36 patients included normal liver function, prior doxorubicin exposure <250 mg/m2, and normal cardiac function. A 1-hour PLD (30 mg/m2) infusion was followed immediately by 1 of 6 trabectedin doses (0.4, 0.6, 0.75, 0.9, 1.1, and 1.3 mg/m2) infused over 3 hours, repeated every 21 days until evidence of complete response (CR), disease progression, or unacceptable txicity. Plasma samples were obtained to assess PK profiles.
The MTD of trabectedin was 1.1 mg/m2. Drug-related grade 3 and 4 toxicities were neutropenia (31%) and elevated transaminases (31%). Six patients responded (1 CR, 5 partial responses), with an overall response rate of 16.7%, and 14 had stable disease >4 months (39%). Neither drug had its PK affected significantly by concomitant administration compared to trabectedin and PLD each given as a single agent.
Trabectedin combined with PLD is generally well tolerated at therapeutic doses of both drugs in pretreated patients with diverse tumor types, and appears to provide clinical benefit. These results support the need for additional studies of this combination in appropriate cancer types.
PMCID: PMC2598415  PMID: 18497430
trabectedin; ET-743; pegylated liposomal doxorubicin (PLD); sarcomas; ovarian cancer
18.  Long-Term Results of Concomitant Boost Radiation plus Concurrent Cisplatin for Advanced Head and Neck Carcinomas: a Phase II Trial of the Radiation Therapy Oncology Group (RTOG 99-14) 
The feasibility of combining concomitant boost accelerated radiation regimen (AFX-C) with cisplatin was previously demonstrated in this phase II trial. This manuscript reports the long-term toxicity, relapse patterns, and survival in patients with advanced head and neck carcinoma (HNC).
Patients and Methods
Between April and November 2000, 84 patients with stage III–IV HNC were enrolled, and 76 patients were analyzable. Radiation consisted of 72 Gy over 6 weeks. Cisplatin dose was 100 mg/m2 on days 1 and 22. Tumor and clinical status were assessed and acute-late toxicities were graded.
The median follow-up for surviving patients is 4.3 years. The 2- and 4-year local-regional failure rates are 33% and 36%, respectively, and the 2- and 4-year survival rates are 70% and 54%, respectively. The worst overall late grade 3 or 4 toxicity rate was 42%. The prevalence rates of a gastrostomy at any time during follow-up, at 12 months, and at 48 months were 83%, 41%, and 17%, respectively. Five of 36 patients (14%) alive and without disease at last follow-up were gastrostomy tube dependent..
These data of long-term follow-up of patients treated with AFX-C with cisplatin show encouraging results with regard to locoregional disease control and survival, with few recurrences after 2 years. The late toxicity rates are relatively high. However, while prolonged dysphagia was noted in our preliminary report, its prevalence does decrease over time. A phase III trial comparing AFX-C plus cisplatin against standard radiation plus cisplatin has completed accrual.
PMCID: PMC2582390  PMID: 18640496
Radiation; chemoradiation; accelerated radiotherapy; late toxicity
19.  Influence of Diabetes on the Interpretation of PET Scans in Patients With Esophageal Cancer 
Patients with diabetes mellitus (DM) can have altered sugar transport into cells, potentially affecting the results of 18-FDG PET scans. The specific aim of this study was to determine the effect of DM on pre- and post-treatment standard uptake value (SUV) scores in patients undergoing chemoradiotherapy for esophageal cancer.
Patients with locally advanced esophageal carcinoma undergoing preoperative or definitive chemoradiotherapy underwent pre- and posttreatment 18-FDG PET scans. Maximum SUV score was measured from the tumor before chemoradiotherapy and 3 to 4 weeks after chemoradiotherapy (preoperatively). Patients were identified as having DM by medical record review. Random serum glucose measurements were obtained prior to 18-FDG PET scans. The Wilcoxon signed-rank test was used to test for differences in SUV scores between patients with and without DM, and a generalized linear model with backward selection was applied to search for significant predictors of initial and posttreatment SUV scores.
Sixty-three patients underwent 18-FDG PET scans during the course of treatment for esophageal malignancies between 6/02 and 8/05. Fifty-four patients received chemotherapy. The median radiation dose was 46.8 Gy. Eighteen patients had DM, six were insulin-dependent DM (IDDM). There was no difference in initial SUV scores between DM and non-DM patients (P > .05). There was also no difference in initial SUV scores between IDDM and non-IDDM groups. Patients with tumors at the gastroesophageal junction had lower initial SUV scores compared to patients with tumors in the lower or mid-esophagus (P = .05). T stage was associated with initial SUV score (T2 lower than T3, P = .014). Older age (P = .03), diabetes (P = .007), higher T stage (P = .002), and presence of nodes (P = .05) were each positively associated with posttreatment SUV scores. Blood glucose levels prior to 18-FDG PET scan, endoscopic tumor length, and tumor location were not predictive of posttreatment SUV scores. Patients with DM had significantly lower posttreatment SUV scores compared to patients without DM (P = .04). Pathologic complete response or percent SUV decrease did not differ between patients with or without DM.
Regardless of glucose levels, DM and IDDM do not influence pretreatment SUV scores in patients with localized esophageal cancer. However, DM may influence posttreatment SUV scores and thus complicate interpretation of treatment response. Further confirmatory study in a larger cohort of DM patients to evaluate the relationship of posttreatment SUV score to pathologic response is warranted.
PMCID: PMC2739639  PMID: 19742140
20.  Review of docetaxel in the treatment of gastric cancer 
Gastric cancer is a global health problem accounting for 800,000 cancer related deaths annually. Often diagnosed at an advanced stage, the treatment of gastric cancer with chemotherapy is directed towards palliating cancer related symptoms with only modest improvements in survival. In addition, no regimen has emerged as a globally accepted standard. New therapeutic options are desperately needed for the treatment of gastric cancer. Docetaxel given in combination has recently emerged as a new option for patients with advanced gastric cancer. This review focuses on the treatment of advanced gastric cancer utilizing docetaxel-based therapy and the novel additions of biotherapy to the existing cytotoxic platforms. In addition, the current investigations of docetaxel for the treatment of potentially curable gastric cancer will be discussed.
PMCID: PMC2621422  PMID: 19209281
docetaxel; gastric cancer; chemotherapy; biotherapy
21.  Thromboembolism in Gastrointestinal Cancers 
The link between thromboembolism and cancer has been recognized for over 100 years. Venous thromboembolism (VTE) is associated with considerable morbidity in patients with cancer, with emerging research also indicating a detrimental effect on survival. Investigations aimed at improving outcomes for patients with cancer have focused on the role of low molecular weight heparin in primary and secondary prevention of VTE and in improving patient survival. Important fundamental questions remain unanswered, however, and a significant line of research needs to be dedicated to investigating VTE in GI cancers. The effect of VTE on survival needs to be clarified, as does the role of anticoagulation in this patient population. Opportunities for additional research include investigating methods to identify patients at risk of developing VTE and developing new strategies and therapeutic interventions to reduce the morbidity and mortality associated with VTE. This review focuses on the current understanding of VTE related to gastrointestinal cancers and directions of interest in research specific to GI cancers and VTE.
PMCID: PMC2632566  PMID: 19259275
22.  Selective Fluorescence Probes for Dipeptidyl Peptidase Activity - Fibroblast Activation Protein and Dipeptidyl Peptidase IV 
Bioconjugate chemistry  2007;18(4):1246-1250.
Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, Fibroblast activation protein (FAP) and Dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion and metastasis. We here validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.
PMCID: PMC2562575  PMID: 17489551
Fibroblast activation protein; dipeptidyl peptidase IV; dipeptidyl peptidase; fluorogenic substrate; fluorescence
23.  Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells 
Molecular and Cellular Biology  2004;24(3):1270-1278.
During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5′ region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2′-deoxycytidine followed by zebularine hindered the remethylation of the p16 5′ region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.
PMCID: PMC321446  PMID: 14729971

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