P225

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS P223. EFFECT OF INITIAL TNM STAGE ON FOLLOWUP STRATEGY FOR RECTAL CANCER PATIENTS. K. Ode 1, K. S. Virgo 2, W. E. Longo 3, R. A. Audisio 1, F. E. Johnson 2; 1University of Liverpool, Liverpool, United Kingdom, 2 Saint Louis University, St. Louis, MO, 3Yale University, New Haven, CT Introduction: For rectal cancer patients, the risk of recurrence after curative-intent treatment is directly related to initial tumor stage. It is commonly assumed that more intensive follow-up is worthwhile in patients with high-stage cancer, while less intensive follow-up is sufficient for those with low-stage lesions. We recently completed a survey of members of the American Society of Colon and Rectal Surgeons (ASCRS) to determine the surveillance strategies they use after primary curative-intent treatment for their own patients. This report focuses on the variation in surveillance intensity ascribable to initial TNM stage. Methods: We created a series of 4 vignettes succinctly describing generally healthy patients with rectal carcinoma (stage I treated with local excision, stage I treated with radical surgery, stage II treated with radical surgery, and stage III treated with radical surgery ⫾ adjuvant therapy). We sent a questionnaire based on the vignettes to all 1,795 members of ASCRS. Evaluable replies were entered into a computer database. The effect of TNM stage on follow-up intensity was analyzed using repeated measures ANOVA. Results: There were 566 responses (32%), among which 347 (61%) were evaluable. The most frequent surveillance modality was office visit. In post-operative year 1 for patients with stage I lesions treated with local therapy, 3.8 ⫾ 1.4 office visits (mean ⫾ SD) were recommended, decreasing to 1.5 ⫾ 0.8 in year 5. For patients with stage III lesions treated with radical surgery ⫾ adjuvant therapy, 4.0 ⫹ 2.8 office visits were recommended in year 1, decreasing to 1.7 ⫾ 1.2 in year 5. Similar results were generated for all commonly used modalities on the questionnaire (3 blood tests, 2 endoscopic procedures, 8 imaging studies). Conclusions: The intensity of post-operative surveillance following curative-intent treatment for rectal cancer varies minimally by TNM stage. Because of this, a randomized trial of alternate follow-up strategies may be feasible without stratification according to stage. We will present the outline of such a trial at the meeting.

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fected with green fluorescence protein (GFP) using a retrovirus. 5⫻10 6 cells were injected subcutaneously in Balb/c nude mice. Once a xenograft reached a minimum volume of 500mm 3, it was harvested and minced for implantation. Using microsurgical techniques, two 1mm 3 pieces of tumor xenograft were subserosally implanted into to 4-6 weeks old Balb/c nude male mice (HCT116 n⫽32 and HCT116b n⫽49). Weekly fluorescence imaging was performed for tumor growth and progression assessment. At 5-9 weeks post implantation, necropsies were performed and tissues procured for histological analysis. Results: All animals in both the HCT116 and HCT116b groups demonstrated local invasion in the colon. Serial fluorescence imaging demonstrated significant primary growth in both groups. However, only the animals implanted with HCT116 demonstrated any significant distant colony formation to either liver and/or lung (Table 1). This difference was statistically significant. Conclusion: This orthotopic model of colon cancer fulfills the rate limiting steps metastasis: invasion and distant colony formation. Both of these isogenic subclones are equally invasive. However, only the HCT116 demonstrates the ability to form distant colonies. Constitutive EGFR activation plays a role within this model system. These two cell lines provide a working in vivo model system to study the biological mechanisms involved in the complex process of metastasis.

Table 1:

HCT 116 HCT 116b

INVASION

LIVER METASTASIS

LUNG METASTASIS

LIVER OR LUNG METASTASIS

100%(32/32) 100%(49/49)

47%(15/32) 4% (2/49)

41%(13/32) 4% (2/49)

60%(19/32) 8% (4/49)

ONCOLOGY XII: CANCER BIOLOGY Figure 1: P224. CHARACTERIZATION OF TWO HUMAN ISOGENIC COLON CANCER SUBCLONES IN AN ORTHOTOPIC MODEL. I. Dominguez 1, R. Rose 1, C. Levea 2, E. Sharratt 3, K. Bullard Dunn 4, M. Brattain 3, A. Rajput 4; 1The State University of New York at Buffalo/Department of Surgery, Buffalo, NY, 2Roswell Park Cancer Institite/Department of Pathology, Buffalo, NY, 3Roswell Park Cancer Institute/Department of Pharmacology & Therapeutics, Buffalo, NY, 4Roswell Park Cancer Institute/Department of Surgical Oncology, Buffalo, NY Introduction: Despite recent medical advances, colon cancer continues as the second leading cause of cancer related deaths in the US with an estimated 55,170 deaths in 2006(ACS). The majority of deaths are related to metastatic disease. In vivo murine models are necessary to study the complex yet poorly understood nature of metastasis. There are two rate limiting steps in metastasis: invasion and distal colony formation. Previously, our group has demonstrated that the human colon cancer isogenic subclones, HCT116 and HCT116b are unique in their in vitro response to growth factor stress deprivation. HCT116 possesses constitutive EGFR activation and therefore is growth factor independent whereas HCT116b lacks constitutive EGFR activation and is growth factor dependent. Purpose: The purpose of this study was to characterize in an in vivo orthotopic model HCT116 and HCT116b cell lines. Methods: Cells were trans-

P225. PLATELET DERIVED GROWTH FACTOR BETA (PDGFR-B) INHIBITION, SUPPRESSES TUMOR GROWTH IN AN ORTHOTOPIC XENOGRAFT MODEL OF EWING’S SARCOMA. A. A. Hayes-Jordan, Y. X. Wang, E. Kleinerman; University of Texas MD Anderson Cancer Center, Houston, TX Background/Purpose: PDGFR-B has been shown to be important in the growth and development of blood vessels. Recently our laboratory has shown that PDGFR-B is found not only in perivascular tissue, but in cell lines and orthotopic xenograft Ewing’s sarcoma tumors. We have previously shown that vascular endothelial growth factor, VEGF, inhibited tumors have increased levels of PDGFR-B and found a significant growth delay but no decrease in incidence of pulmonary metastasis in an orthotopic xenograft model of Ewing’s sarcoma. The purpose of this study is to evaluate the importance of PDGFR-beta in the growth and metastasis of Ewing’s sarcoma. Methods: We used small interfering RNA, siRNA, to reduce PDGFR-beta in a human Ewing’s sarcoma cell line, TC-71. Four different sequences were tested representing different portions of the PDGFR-B gene. One clone was chosen. In our orthotopic xenograft model, these TC-71 cells were injected into the periosteum of the ribs of ‘nude’ mice. We compared the growth of tumors after injection of TC-71, TC-71 vector, and TC-71pd cells into this model. Results:

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

One clone, TC-71pd showed 90% reduction in PDGFR-Beta thus verifying successful reduction by siRNA. In this model, we have consistently seen a 60-70% incidence of chest wall tumors and no pulmonary metastasis and 20-30% incidence of pulmonary metastasis and little or no chest wall tumor. As expected in the TC-71 group, 70% (7/10) of mice had chest wall tumors and 20%(2) mice had pulmonary metastasis. In the TC-71vector group, 70%(7/10)and 60% (6/10)o f the mice in the TC-71pd group grew chest wall tumors. None of the mice in the TC-71pd or vector group developed pulmonary metastasis. On day 14 after injection, the average size of the TC71pd tumors were 110mm 3, TC-71 vector, 223mm 3 and the TC-71 342mm 3. On day 21 after injection the average tumor size was 286mm 3 in the TC-71pd, 590mm 3 in the TC-71 vector control, and 770 mm 3 in the TC-71 tumors. (p⫽0.028). Conclusions: Inhibition of PDGFR-beta, significantly decreased tumor size and suppressed tumor growth rate in an orthotopic model of Ewing’s sarcoma. The incidence of chest wall tumors and spontaneous lung metastasis was not significantly different in tumors grown from the PDGFR-beta inhibited cell line, TC-71pd, compared to vector control. This may be because other angiogenic factors such as vascular endothelial growth factor, VEGF, remain at normal levels. Because of the significant reduction in tumor size seen in this model, PDGFR-beta should be further studied in the context of combination therapy in the treatment of Ewing’s sarcoma.

P226. CYCLOOXYGENASE-2 EXPRESSION INDUCES GENOMIC INSTABILITY IN MCF10A BREAST EPITHELIAL CELLS. L. E. Vincent, B. Singh, J. A. Berry, A. S. Multani, A. Lucci, Jr.; The University of Texas MD Anderson Cancer Center, Houston, TX Introduction: COX-2 is induced in many breast cancers, and COX-2 expression correlates with a worse outcome in the clinic. We hypothesized that COX-2 positive breast cancers develop additional genomic alterations that can no longer be reversed with a COX-2 inhibitor, and the induction of genomic instability is a major mechanism through which COX-2 contributes to cancer progression. Methods: We co-transfected MCF10A nonmalignant immortalized breast epithelial cell line with a pSG5COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). The control cells were transfected with the luciferase vector alone. We confirmed COX-2 expression by western blot, and its functionality by measuring PGE 2 (a product of the COX-2 pathway) production by an immunoassay. We analyzed the genomic instability phenotype by chromosome analysis of control and COX-2 transfected metaphase-arrested MCF10A cells after Giemsa staining. To understand the molecular basis of COX-2 mediated genomic instability, we carried out a microarray analysis on COX-2 transfected and control MCF10A cells. Groups were compared using chi-square tests. Results: Cytogenetic analysis of early passage COX-2 transfected cells showed that COX-2 expression increased genomic instability as compared to MCF10A cells transfected with a luciferase vector alone. COX-2 overexpression was associated with a significant increase in chromosomal aberrations (fusions, breaks, and tetraploidy; see Table). There was a statistically significant increase in the number of polyploid cells in the COX-2 transfected group versus control (p⫽0.004). We observed gene expression changes associated with COX-2 overexpression (including an increase in HDM2 and cyclin E1, and decrease in p57 kip2 ) indicating that that COX-2 transfection caused not only genomic instability (an important early step in tumorigenesis) but also abnormal cell cycle regulation. Further characterization of the pre-malignant phenotype of COX-2 transfected MCF10A cells showed that COX-2 expression confers significant changes in cell morphology and growth characteristics, and confers resistance to anoikis. Maintenance of the pre-malignant phenotype was confirmed through inability of the COX-2 transfected MCF10A cells to establish tumors in a nude mouse model of

malignancy. Conclusions: We found that COX-2 expression in MCF10A breast epithelial cells confers a pre-malignant phenotype that includes enhanced genomic instability, and altered cellcycle regulation.

Chromosome analysis by Giemsa staining of metaphase-arrested cells

Sample

No. of cells analyzed

% normal diploid cells

% cells with aberrations

% cells with fusions

% polyploid cells

MCF 10A MCF 10A COX-2

35 40

94.3 67.5

0 12.5

0 5

5.7 22.5

P227. ANTERIOR GRADIENT 2 AND TFF1 COEXPRESSION IN BREAST CANCER LYMPH NODE METASTASES FROM ESTROGEN RECEPTOR-NEGATIVE (ER-) PRIMARY TUMORS CONTRIBUTES TO THE METASTATIC PHENOTYPE. P. Davoodi, A. Graham, K. Mikhitarian, M. Mitas, D. J. Cole; Medical University of South Carolina, Charleston, SC A more complete understanding of the critical genes regulating the phenotypic characteristics associated with the development of metastatic breast cancer could help impact on the effectiveness of therapy for this disease. In order to identify target genes involved in breast cancer metastasis, we performed a microarray analysis of three metastatic lymph nodes. Thirty-five genes that were most highly expressed as compared to negative control cervical lymph nodes were identifyed. After cytokeratin 19, the second most highly expressed gene was anterior gradient 2 (AGR2), a gene known to be upregulated in estrogen receptor positive (ER⫹) tumors. We next wanted to determine the relative contribution of this gene’s over expression to the metastatic phenotype. To approach this, we measured by quantitative real-time RT-PCR the expression levels of AGR2 and 15 other breast cancer-associated genes in pathology-positive axillary lymph nodes (ALN) derived from ER⫹ (n⫽53) and ER- (n⫽17) primary tumors. Of the 15 genes analyzed, we observed that expression of AGR2 was most highly correlated with TFF1 expression levels,, a known estrogeninducible gene. Furthermore, the correlation between AGR2 and TFF1 was essentially the same whether ALN was derived from ER⫹ or ER- primary tumors (R 2 ⫽ 0.81 and 0.89, respectively). The unexpected correlation between two ER-associated genes in ER- breast cancer cells suggested to us that co-expression of AGR2 and TFF1 may be contributory to the metastastic phenotype of ER- cells. To investigate this possibility, we used an ER- cell line (MDA453) that expressed the ER gene at a level 10 -2 times less than the ER⫹ cell line MCF7. Transfection of MDA453 with siRNA directed against AGR2, TFF1, or a combination of the two, resulted in a 28.4% ⫹ 4%, 32.02% ⫾ 6%, and 57.05% ⫾ 2% (respectively), reduction in cell invasion. Transfection of AGR2 or TFF1 siRNA also caused an unexpected 40-fold reduction in ER mRNA levels as determined by quantitative real-time RT-PCR measurements. Our results provide evidence that expression of AGR2 and TFF1 in ER-negative tissues contribute to the metastatic phenotype. Further work is ongoing to define the mechanisms involved, which could include transcriptional activation of genes in the ER pathway. P228. GEMCITABINE RESISTANCE OF PANCREATIC TUMOR CELLS IN CULTURE IS ASSOCIATED WITH PROPERTIES OF EPITHELIAL TO MESENCHYMAL TRANSITION. A. N. Shah, G. Gallick; MD Anderson Cancer Center, Houston, TX Introduction: Pancreatic cancer afflicts over 33,000 people in the United States yearly and has an extremely poor prognosis. The five-year survival rate stands at less than 5%, due largely to