Faculty Publications

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 a fluorescent photosensitizer and a black hole quencher 3 linked by a peptide sequence (TSGPNQEQK) specific to FAR FAP-PPB was effectively cleaved by both human FAP and murine FAR By use of 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.

In the calculation of pointflux by Monte Carlo simulation, there is a special disadvantage in the mostly used method of next event estimation (NEE) for which theoretical variance is infinite. And, this problem has not yet been solved satisfactorily. The purpose of this paper is to provide some new ideas to solve the problem of infinite variance without introducing any bias for the mean. To eliminate the unbounded factors, the relations among the different state variables for two neighboring collisions are analyzed; then, on the basis of the integral expression of the once-more scattered flux contribution to the point detector, by changing the state variables to be sampled, six basic methods are derived-two of them are NEE and collision probability estimation, and four are new methods. Furthermore, based on one of the new methods, by variable substitution, a new method called exponent biased sample estimation (EBS) is obtained that can eliminate the vertical bar(r) over right arrow (d) - (r) over right arrow (m)vertical bar(-2) singularity factor and has no exponent factor, which exists in other methods. The benchmark results show that EBS is much better than NEE with the variance of one order of magnitude smaller and a figure-of-merit factor of several hundreds higher sometimes, and its calculation efficiency is higher than that of the once more collision flux estimation method Compared with the direction biased sample estimation method, EBS has no advantage in variance, but the sample procedures are much simpler and use less CPU time.

OBJECTIVES: Given the extensive desmoplastic response associated with pancreatic adenocarcinoma, we hypothesized that the stromal protein fibroblast activation protein (FAP) would be highly expressed and associated with the presence of fibrosis and other clinical features. METHODS: Paraffin-embedded pancreatic adenocarcinomas that were resected with curative intent from 1992 to 2003 were used for this study. Fibroblast activation protein expression by immunohistochemical analysis was evaluated both by intensity (0-3+) and percentage. Fibrosis was estimated as a percentage of each tumor specimen. RESULTS: Ninety percent (63/70) of specimens demonstrated FAP expression. Expression was significantly more pronounced in tumor-associated myofibroblasts immediately adjacent to tumor than in surrounding tumor-associated myofibroblasts (P < 0.001). Lower FAP expression in adjacent tumor-associated myofibroblasts was associated with increased fibrosis (P = 0.02). Greater FAP expression in surrounding tumor-associated myofibroblasts was associated with an increased chance of having positive lymph nodes for all patients (P = 0.03) and a higher risk of tumor recurrence (P = 0.015) and death (P = 0.02) for patients who did not receive preoperative therapy. CONCLUSIONS: Fibroblast activation protein is highly expressed in pancreatic adenocarcinoma, with greatest expression immediately adjacent to tumor. Higher FAP expression is associated with worse clinical outcome. The investigation of FAP inhibitors as a therapeutic strategy against pancreatic cancer should be considered.

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.

BACKGROUND: 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/m(2), and normal cardiac function. A 1-h PLD (30 mg/m(2)) infusion was followed immediately by one of six trabectedin doses (0.4, 0.6, 0.75, 0.9, 1.1, and 1.3 mg/m(2)) infused over 3 h, repeated every 21 days until evidence of complete response (CR), disease progression, or unacceptable toxicity. Plasma samples were obtained to assess PK profiles. RESULTS: The MTD of trabectedin was 1.1 mg/m(2). Drug-related grade 3 and 4 toxic effects were neutropenia (31%) and elevated transaminases (31%). Six patients responded (one CR, five partial responses), with an overall response rate of 16.7%, and 14 had stable disease (less than a 50% reduction and less than a 25% increase in the sum of the products of two perpendicular diameters of all measured lesions and the appearance of no new lesions) >4 months (39%). Neither drug had its PK affected significantly by concomitant administration compared with trabectedin and PLD each given as a single agent. CONCLUSION: 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.

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. Here, we 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.

PURPOSE: Human fibroblast activation protein (FAP)/seprase is a 97-kDa surface glycoprotein expressed on tumor associated fibroblasts in the majority of epithelial cancers including colon adenocarcinomas. FAP overexpression in human tumor cells has been shown to promote tumor growth in animal models, and clinical trials targeting FAP enzymatic activity have been initiated. The primary objective of this study was to evaluate the clinical significance of stromal FAP in human colon cancers by immunohistochemisty. EXPERIMENTAL DESIGN: Sections of paraffin-embedded resected primary human colon cancer specimens from 1996 through 2001 within the Fox Chase Cancer Center tumor bank were stained with D8 antibody directed against FAP/seprase. Xenotransplanted human colorectal tumors in mice were examined similarly for stromal FAP in tumors of different sizes. Overall percentage of stromal FAP staining of the primary tumor was assessed semiquantitatively (0, 1+, 2+, 3+) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. RESULTS: One hundred thirty-eight patients with resected specimens were available for study (mean follow-up, 1,050 days) with 6 (4%) stage I, 52 (38%) stage II, 43 (31%) stage III, and 37 (27%) stage IV patients. FAP was detected in >93% of specimens. Semiquantitative staining was scored as 1+ in 28 (20%), 2+ in 52 (38%), and 3+ in 49 (35%). FAP staining intensity was graded as weak in 45 (33%), intermediate in 48 (35%), and dark in 36 (26%). Stromal FAP was found to correlate inversely with tumor stage (semiquantitative, P = 0.01; intensity, P = 0.009) and with tumor size of the tumor xenograft model (correlation coefficient, -0.61; P = 0.047), suggesting that stromal FAP may have a greater role in the early development of tumors. Furthermore, greater stromal FAP for patients with known metastatic disease was associated with a decreased survival. CONCLUSION: Our data indicate that patients whose colon tumors have high levels of stromal FAP are more likely to have aggressive disease progression and potential development of metastases or recurrence. This study affirms the rationale for ongoing clinical investigations using FAP as a therapeutic target to disrupt FAP-driven tumor progression in patients with metastatic disease. It also suggests that the effects of FAP inhibition should be investigated in earlier-stage tumors, given its high levels and potential effect earlier in the course of the disease.

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene have frequently been detected not only in schwannomas and other central nervous system tumors of NF2 patients but also in their sporadic counterparts and malignant tumors unrelated to the NF2 syndrome such as malignant mesothelioma, indicating a broader role for the NF2 gene in human tumorigenesis. However, the mechanisms by which the NF2 product, merlin or schwannomin, is regulated and controls cell proliferation remain elusive. Here, we identify a novel GTP-binding protein, dubbed NGB (referring to NF2-associated GTP binding protein), which binds to merlin. NGB is highly conserved between Saccharomyces cerevisiae, Caenorhabditis elegans, and human cells, and its GTP-binding region is very similar to those found in R-ras and Rap2. However, ectopic expression of NGB inhibits cell growth, cell aggregation, and tumorigenicity in tumorigenic schwanomma cells. Down-regulation and infrequent mutation of NGB were detected in human glioma cell lines and primary tumors. The interaction of NGB with merlin impairs the turnover of merlin, yet merlin does not affect the GTPase nor GTP-binding activity of NGB. Finally, the tumor suppressor functions of NGB require merlin and are linked to its ability to suppress cyclin D1 expression. Collectively, these findings indicate that NGB is a tumor suppressor that regulates and requires merlin to suppress cell proliferation.

Purpose: Development of targeted therapeutic agents in colorectal cancer (CRC) is impeded by the lack of a noninvasive surrogate of drug effect. This pilot study evaluated the ability of immunomagnetic separation to isolate circulating tumor cells (CTCs) and of the fluorescent microscope system and flow cytometry to enumerate and characterize CTCs from patients with metastatic CRC. Patients and Methods: Fifty patients with metastatic CRC contributed 50 mL of blood at treatment initiation and disease evaluation timepoints. Fresh tumor specimens were obtained from 17 patients for comparison of circulating and in situ tumor cell characteristics. Epithelial cells were magnetically isolated from whole blood targeting the antiepithelial cell adhesion molecule (EpCAM). Circulating tumor cells were defined as EpCAM isolated, cytokeratin positive, nuclear stain positive, and CD45 negative. Total RNA was isolated from EpCAM-enriched CTCs and multigene reverse-transcriptase polymerase chain reaction analyses were performed. Results: The median number of CTCs detected by flow cytometry was 2/7.5 mL blood. Mean change in cell count was significantly different for patients with tumor progression versus nonprogression (+6.7 vs. +0.2/7.5 mL; P = 0.001). A correlation was noted between mean fluorescence intensity (flow cytometry) of cytokeratin in CTC and matched tumor specimens (r = 0.79, P = 0.06). Nearly 80% (15 of 19) of samples with >= 2 CTCs expressed >= 1 epithelial marker gene (CK19, CK20, carcinoembryonic antigen, or epidermal growth factor receptor). Conclusion: Isolating and characterizing CTCs from patients with metastatic CRC is feasible. Change in the CTC number might reflect clinical status, and flow cytometric and gene expression data suggest similarity of circulating and in situ tumor cells. Further evaluation of CTCs for pharmacodynamic and clinical monitoring in patients with CRC is warranted.

Background: Multimodality therapy for esophageal cancer holds promise for improving outcome in this lethal disease. On the basis of encouraging data from a phase I trial, we conducted a phase II study of preoperative chemotherapy, followed by concurrent chemoradiotherapy and surgery. Methods: Patients with clinically staged resectable esophageal cancer were treated with induction cisplatin and paclitaxel, followed by 45 Gy of external beam radiation with concurrent infusional 5-fluorouracil and weekly cisplatin and paclitaxel. Four to eight weeks after multimodality induction, esophagectomy was performed in suitable patients. Study end points were survival, pathologic complete response, and toxicity. Results: Twenty-one patients were enrolled with a median age of 58 years, and all patients were clinically staged II or III. Sixteen (76.2%) patients completed the trial, of whom four (25%) had a pathologic complete response. One patient died from postoperative complications. Grade 3 or 4 toxicity was observed in 76% of patients, and dose-limiting toxicity was seen in 6 of the first 14 patients, thus necessitating a planned dose reduction of paclitaxel. At a median follow-up of 30 months, 13 patients remain alive. The 2-year disease-specific survival for the study population was 78%. Conclusions: This regimen of multimodality therapy before resection resulted in an encouraging 2-year survival rate but a disappointing rate of pathologic complete response and was toxic, necessitating a predetermined paclitaxel dose reduction. The incorporation of taxanes into induction strategies for esophageal cancer seems promising, but the optimal schedule remains undefined.

SO JOURNAL OF CLINICAL ONCOLOGY LA English DT Meeting Abstract C1 Fox Chase Canc Ctr, Philadelphia, PA 19111 USA. Univ Oklahoma, Hlth Sci Ctr, Oklahoma City, OK USA

Purpose To investigate the feasibility of combining concomitant boost accelerated radiation regimen (AFX-C) with cisplatin and to assess its toxicity and the relapse pattern and, survival in patients with advanced head and neck carcinoma (HNC). Patients and Methods Between April and November of 2000, 84 patients with stage III to IV HNC who met the eligibility criteria were enrolled; 76 of these patients were analyzable. Radiation consisted of 72 Gy in 42 fractions over 6 weeks (daily for 3.5 weeks, then twice a day for 2.5 weeks). Cisplatin dose was 100 mg/m(2) on days 1 and 22. Tumor and clinical status were assessed, and acute late toxicities were graded. Results Sixty-five patients (86%) received both radiation and chemotherapy per protocol or with minor variations. The estimated 2-year locoregional relapse and distant metastasis rates were 34.7% and 16.1%, respectively. The estimated 2-year overall survival and disease-free survival rates were 71.6% and 53.5%, respectively. Three patients (4%) died of complications, 19 patients (25%) had acute grade 4 toxicity, and 49 patients (64%) had acute grade 3 toxicity. The 2-year cumulative incidence of late grade 3 to 5 toxicities was 5.1.3%. Conclusion These data showed that it was feasible to combine AFX-C with cisplatin. The compliance to therapy was high, and the locoregional control and survival rates achieved compared favorably with AFX-C alone or other concurrent chemoradiation regimens tested by the Radiation Therapy Oncology Group. A phase III trial comparing AFX-C plus cisplatin against standard radiation plus cisplatin is ongoing to determine whether the use of AFX-C in the concurrent chemoradiation setting further improves outcome.

Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting its enzymatic activity.