Faculty Publications

OBJECTIVES: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. METHODS: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS). RESULTS: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA). CONCLUSIONS: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 microL of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.

During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

BACKGROUND: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. METHODS: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. RESULTS: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7, respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. CONCLUSIONS: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.

We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

BACKGROUND: Cetuximab has demonstrated synergy with taxanes in preclinical models; as well as single agent activity. We assessed the activity of cetuximab with carboplatin and paclitaxel given on a 4-week schedule, in advanced, chemo-naive non-small cell lung cancer. PATIENTS AND METHODS: This phase II, single arm, multi-institution study featured standard dosage of cetuximab 400 mg/m day 1, then 250 mg/m with paclitaxel (100 mg/m/wk, for 3 weeks), and carboplatin (area under curve = 6) day 1 of each 28 day cycle. After 4 to 6 cycles, in the absence of disease progression or excess toxicity, cetuximab was continued weekly. Primary end point was response rate. RESULTS: Fifty-three patients (median age 63, 51% male) participated. Response rate was 57% (3 complete response and 27 partial response). At a median follow-up of 12.5 months, the estimated overall survival is 13.8 months (95% CI: 9.08-16.02) with an event-free survival rate of 5.53 months (95% CI: 4.77-7.99), 18.9% remain free from progression at 1 year. Improved survival was associated with female gender, absence of prior radiation, PS 0 and epidermal growth factor receptor expression. Toxicities included rash (28% grade 3), nail changes (3.7% grade 3), hypomagnesemia (7.5% grade 3 and 3.7% grade 4), and neutropenia (25% grade 3 and 13% grade 4) in addition to other typical side effects anticipated with paclitaxel/carboplatin. There were no grade 5 toxicities. CONCLUSION: Combination of cetuximab/paclitaxel/carboplatin in non-small cell lung cancer was well tolerated and clinically active with manageable toxicities. This unique schedule, integrating weekly paclitaxel and cetuximab has not yet been tested in a randomized trial.

Telomere attrition ultimately leads to the activation of protective cellular responses, such as apoptosis or senescence. Impairment of such mechanisms can allow continued proliferation despite the presence of dysfunctional telomeres. Under such conditions, high levels of genome instability are often engendered. Data from both mouse and human model systems indicate that a period of genome instability might facilitate tumorigenesis. Here, we use a liposarcoma model system to assay telomere maintenance mechanism (TMM)-specific genetic alterations. A multiassay approach was used to assess the TMMs active in tumors. Genomic DNA from these samples was then analyzed by high-resolution DNA mapping array to identify genetic alterations. Our data reveal a higher level of genome instability in alternative lengthening of telomere (ALT)-positive tumors compared with telomerase-positive tumors, whereas tumors lacking both mechanisms have relatively low levels of genome instability. The!

The mammalian target of rapamycin (mTOR) is thought to play a critical role in regulating cell growth, cell cycle progression, and tumorigenesis. Because the AKT-mTOR pathway is frequently hyperactivated in ovarian cancer, we hypothesized that the mTOR inhibitor RAD001 (Everolimus) would inhibit ovarian tumorigenesis in transgenic mice that spontaneously develop ovarian carcinomas. We used TgMISIIR-TAg transgenic mice, which develop bilateral ovarian serous adenocarcinomas accompanied by ascites and peritoneal dissemination. Fifty-eight female TgMISIIR-TAg mice were treated with 5 mg/kg RAD001 or placebo twice weekly from 5 to 20 weeks of age. To monitor tumor development, mice were examined biweekly using magnetic resonance microimaging. In vivo effects of RAD001 on Akt-mTOR signaling, tumor cell proliferation, and blood vessel area were analyzed by immunohistochemistry and Western blot analysis. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by approximately 84%. In addition, ascites formation, together with peritoneal dissemination, was detected in only 21% of RAD001-treated mice compared with 74% in placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. Treatment with RAD001 diminished the expression of vascular endothelial growth factor in tumor-derived cell lines and inhibited angiogenesis in vivo. RAD001 also attenuated the expression of matrix metalloproteinase-2 and inhibited the invasiveness of tumor-derived cells. Taken together, these preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer. [Cancer Res 2007;67(6):2408-13].

BACKGROUND:: Irinotecan and cisplatin individually are active in non-small cell lung carcinoma (NSCLC). Each is synergistic with radiation. Dosages of 65 mg/m of irinotecan and 30 mg/m of cisplatin Q weekly times four every 6 weeks yielded a 36% response rate and median survival of 11.6 months in advanced NSCLC (Jagasia et al.; Clinical Cancer Reserch 7: 68, 2001). A weekly schedule for each agent (versus less frequent doses) limits toxicity and increases the opportunity for radiosensitization. MATERIALS AND METHODS:: We initiated a phase I study of weekly irinotecan and cisplatin during radical thoracic radiation (TRT). Cisplatin was fixed at 25 mg/m Q weekly times seven. Irinotecan was dosed initially at 30 mg/m per week for 7 weeks and was increased by 10 mg/m per week in three- to six-patient cohorts. TRT was administered in 34 single daily fractions to 63 Gy. Eligibility stipulated locally advanced NSCLC; Eastern Cooperative Oncology Group performance status 0 to 1; </=10% unintended weight loss; and adequate physiologic indices. RESULTS:: Fifteen patients were accrued: nine were stage IIIB, five were stage IIIA, and one had isolated mediastinal node recurrence after prior surgery. Median age was 65 years (range, 47-77). Seven patients received irinotecan at a dose of 30 mg/m per week; (dose level 1). Seven other patients received irinotecan at a dose of 40 mg/m per week; (dose level 2). The one other patient received irinotecan in doses of 50 mg/m per week; (dose level 3). Neutropenic fever occurred in one patient each at dose levels 1 and 2. Grade 4 neutropenia occurred in three patients at each dose level. Transient grade 3 diarrhea occurred in one patient at dose level 1. Esophagitis of grade 3 or higher occurred in one patient each at dose levels 2 and 3. There was one late grade 3 pneumonitis at dose level 2. Delivered irinotecan dose intensity for dose level 1 was 27 mg/m per week; for dose level 2, it was 31.4 mg/m per week. Nine of 13 evaluable patients (69%) responded. At median potential follow-up of 5 years, 14 have progressed, and 11 have died. Projected median survival is 28 months; one patient who was treated for mediastinal node recurrence remains free from progression at 6 years. CONCLUSION:: Weekly irinotecan and cisplatin combined with radical TRT (63 Gy) is active and fairly well tolerated in locally advanced NSCLC. In combination with fixed-dose cisplatin (25 mg/m per week), the maximum-tolerated dose of irinotecan is 30 mg/m per week.

Our laboratory developed a transgenic mouse model of spontaneous epithelial ovarian cancer (EOC) in which tumors are initiated by expression of the early region of the Simian Virus 40 (SV40) under transcriptional control of the 5' upstream regulatory region of the Mullerian inhibiting substance type 11 receptor (MISIIR) gene. Female TgMISIIR-TAg-DR26 transgenic mice develop bilateral ovarian tumors with variable latency and survive an average of 152 days. In the absence of reliable methods for disease detection and evaluation of therapeutic response, preclinical studies of this transgenic mouse model of EOC would be limited to longitudinal experiments involving large numbers of animals with euthanasia as the endpoint. Therefore, a non-invasive method for detecting tumors, measuring tumor volume and calculating parameters relevant to the evaluation of therapeutic or preventive interventions (i.e., tumor growth rates, tumor initiation, tumor regression and the time for tumors to reach a given size) is required. We developed and optimized a non-invasive Magnetic Resonance Imaging (MRI) scanning protocol to obtain high resolution abdominal images that is well tolerated by mice. Superior contrast and contrast to noise ratio (CNR) was found with Gd-DTPA contrast enhanced T-1-weighted sequences. Image sets in both the axial and coronal orientations for redundant measurements of normal ovary and ovarian tumor volume can be acquired in approximately 20 minutes. Accuracy of MRI-based ovary and tumor volume determinations was verified by standard volume measurements at necropsy. Serial imaging studies were performed on 41 ovarian cancer bearing TgMISIIR-TAg-DR26 transgenic mice to quantitate tumor progression over time in this model. A chemotherapy study was conducted on TgMISIIR-TAg-DR26 transgenic mice using a standard combination therapy consisting of cisplatin and paclitaxel. Our results demonstrate that MRI is well tolerated and can be repeated in serial imaging studies, permitting quantitative analysis of tumor growth and progression and response to therapeutic interventions.

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

Hepatocyte turnover appears to be an important feature in the resolution of transient and progression of chronic hepadnavirus infections. Hepatocyte death, initiated through attack by antiviral cytotoxic T-lymphocytes (CTL), and compensatory hepatocyte proliferation, are both believed to be major contributing factors in the loss of virus DNA during immune resolution of transient infections. Noncytopathic curing of hepatocytes is also suggested to occur, though this mechanism does not prevent the death of large numbers of hepatocytes. Hepatocyte death, proliferation and curing are also important features of chronic infections, though the outcomes are different. In particular, immune selection due to persistent attack by antiviral CTL is thought to play a role in the emergence of hepatocytes infected with mutant strains of hepatitis B virus (HBV) (e.g. HBV e antigen-negative strains) and in the emergence of hepatocytes that appear refractory to HBV infection. In both instances, clonal expansion of subpopulations of hepatocytes may be inferred to have taken place. Interestingly, foci of altered hepatocytes and hepatocellular carcinomas (HCC) typically do no support virus replication. Thus, immune selection of hepatocytes by antiviral CTL, by inducing clonal expansion, may also play an important role in the progression to HCC. In this review, we discuss the evidence in support of roles for hepatocyte turnover in the resolution of transient and progression of chronic HBV infections.