Intranodal immunization with a dendritic cell tumorvaccine is more effective than subcutaneous orintravenous routesLaura A Lambert, M.D., Glen R Gibson, M.D., Richard J Barth, Jr.,M.D, FACS. Dartmouth-Hitchcock Medical Center, 1 Medical CenterDrive, Lebanon, NH 03756, USA; Contact person: Richard J. Barth,Jr., M.D., FACS Tel. (603) 650-7903

Introduction: In murine tumor models, tumor antigen-pulsed dendriticcells (DCs) administered subcutaneously (SC) can generate anti-tumorimmune responses. However, only a small fraction of SC injected DCsmigrate to regional lymph nodes and activate T-lymphocytes. There-fore, we compared the potency of 3 different routes of administrationof a murine tumor lysate-pulsed DC vaccine: SC, intravenous (IV) anddirectly into a lymph node (LN).

Methods: Groups of 8 mice were immunized via SC, IV or LN injectionof DCs pulsed with MCA-105 sarcoma or TS/A adenocarcinoma tumorlysate. On day 7, pulmonary metastases were established via tail-veininjections. On day 21, the mice were sacrificed and the mean numberof pulmonary metastases per group were compared using theWilcoxon-Rank Sum test.

Results: As shown in Figure 1, LN immunization was approximately4-fold more effective at suppress-ing the development of pulmonarymetastases than SC (p 5 0.04).LN injection of a DC vaccine wasalso more effective than IV (meannumber of pulmonary metastases29 vs 71, p 5 0.01). Similar resultswere obtained in both tumor mod-els. Antigen specific proliferationof spleen derived CD4 and CD8 Tcells was 10-fold higher in miceimmunized LN compared with SC.

Conclusions: Immunization via intranodal injection of DC vaccines ismore efficacious than SC or IV immunizations. This has implications forDC tumor vaccine trials in patients.

Adoptive immunotherapy augmented withcyclophosphamide (CY) plus interleukin-12 (IL-12) caneradicate large subcutaneous MCA207 tumors in Tcell receptor-b knockout mice (TCRb-KO)Hop N Le, MD, Kangla Tsung, PhD, Jeffrey A Norton, MD, FACS.UCSF/SFVAMC, Surgical Service 112, San Francisco VA MedicalCenter, 4150 Clement Street, San Francisco, CA, 94121, USA;Phone: (415)221-4810 ext. 2962; Fax: (415)750-2181.

Introduction: Tumor reactive T cells used for systemic adoptive im-munotherapy have no immediate or overt capacity of their own tocause tumor regression. The challenge of adoptive immunotherapy isto find ways to nurture these T cells following transfer. We havedeveloped an adoptive immunotherapy model in which systemic trans-fer of subtherapeutic numbers of tumor reactive spleen cells into largesubcutaneous (s.c.) MCA207 tumor bearing T cell receptor-b knockout(TCRb-ko) mice can result in complete tumor eradication with Cy 1IL-12 treatment.

Methods: Tumor reactive spleen cells were harvested from C57BL/6mice cured of large s.c. MCA207 tumors with Cy 1 IL-12 treatmentC57BL/6-TCRb-ko mice received systemic i.v. adoptive spleen celltransfer 14 days after s.c. tumor inoculation with 5 3 105 MCA207tumor cells. Treatment occurs seven days after adoptive transfer withCy (3mg/mouse i.p.) followed four days later with recombinant murineIL-12 (200ng i.p. every other day for 3 doses).

Results: TCRb-ko mice cannot be cured of s.c. MCA207 tumor withCy1IL12 treatment (0/5) or with systemic adoptive transfer of tumorreactive spleen cells (0/3) alone. However, Cy 1 IL-12 treatment ofMCA207 tumor bearing TCRb-ko mice seven days after adoptivetransfer with tumor sensitized spleen cells resulted in complete erad-ication of the tumor (3/3). Cy 1 IL-12 treatment cannot cure tumorbearing TCRb-ko mice that received adoptive transfer with naïvespleen cells (0/3). Subtherapeutic numbers of tumor sensitized spleen

cells as low as 1/16 of a spleen can result in complete tumor eradica-tion after CF 1 IL-12 treatment.

Conclusions: Treatment with Cy 1 IL-12 after systemic adoptivetransfer of subtherapeutic numbers of tumor sensitized T cells caneradicate large s.c. MCA207 tumors in TCRb-ko mice.

Additive reduction of intestinal neoplasia with APCgene replacement and COX-2 inhibitionJohn I Lew, M.D., Richard K Kim, B.A., Yuee Guo, M.S., FabrizioMichelassi, M.D., F.A.C.S., Richard B Arenas, M.D., F.A.C.S.Department of Surgery, The University of Chicago Pritzker School ofMedicine, Chicago, IL; Contact: J.I. Lew, M.D., Department ofSurgery, The University Chicago, 5841 S. Maryland Avenue, MC5031, Chicago, IL 60637, USA. (773) 702-2048; Fax (773) 702-0564

Introduction: Multiple Intestinal Neoplasia (Min) mice, heterozygousfor a mutation of the APC gene, develop neoplastic intestinal polypswith elevated cyclooxygenase-2 (COX-2) levels similar to patients withcolorectal cancer. Liposomal delivery of human APC gene may preventor reduce intestinal neoplasia. Furthermore, increased COX-2 levelsfound in colon cancer provide a rationale for the early use of selectiveCOX-2 inhibitors such as Vioxx to prevent polyp formation. This studywas performed to determine whether liposomal tumor suppressor APCgene therapy and further selective COX-2 inhibition with Vioxx mighthave an additive effect in the reduction of intestinal neoplasia in theMin mouse.

Methods: Five-week old C57BL/6J-Min/1 mice were weaned onto ahigh fat (30%) diet with or without Vioxx. The animals were randomizedinto four treatment groups (n 5 10 for each group): control, APC only,Vioxx only and combined APC/Vioxx. For the APC treated groups,plasmid (20mg) containing human APC gene with a viral cytomegalo-virus promoter (pCMV-APC) mixed with lipofectin (200ml) was admin-istered transorally bi-weekly. Vioxx was administered orally to the miceat 200 p.p.m. in the high fat diet. Control mice were treated with aplasmid lacking APC (pCMV-neo) in an identical fashion. After twomonths, the mice were sacrificed and intestinal polyps were countedand measured in size.

Results: Examination of small intestine and colon demonstrated asignificant reduction in the overall total number of intestinal polyps inthe APC and Vioxx treatment groups, especially in the distal smallbowel where polyp formation was greatest. There was a decrease inoverall polyp size in the APC and Vioxx treatment groups, although notsignificant. Western blot analysis showed higher levels of normal APCprotein expression in the repetitively APC treated mice.

Treatment

ProximalSmall Bowel

(polyps/mouse)

Distal SmallBowel

(polyps/mouse)

Colon(polyps/mouse)

Total(polyps/mouse)

Polypsize (mm)

Control 15.1 6 1.8 43.3 6 5.8 1.8 6 0.4 60.2 6 7.1 1.6 6 0.2APC 9.7 6 1.3* 17.1 6 2.5** 0.7 6 0.2* 27.8 6 3.4** 1.3 6 0.04Vioxx 6.2 6 1.3** 10.9 6 2.8** 0.7 6 0.3* 17.8 6 4.2** 1.0 6 0.2APC/Vioxx 3.1 6 0.7** 4.5 6 1.1** 0.3 6 0.2* 7.9 6 1.1** 1.0 6 0.2

All data are expressed as mean 6 SEM. * P # 0.05 vs. pCMV-neo (control) byANOVA. ** P # 0.005 vs. pCMV-neo (control) by ANOVA.

Conclusions: APC gene replacement and COX-2 inhibition have anadditive effect in the prevention of polyp formation as shown in the Minmouse, thereby suggesting their respective roles in colorectal tumor-igenesis as two independent pathways. Each modality in itself mayprove therapeutic and contribute to new strategies in the treatment ofcolon cancer.

Antitumor efficacy of a cytokine secreting, oncolyticherpes simplex virusSandeep Malhotra MD, Joseph Bennett MD, Jonathan Zager MD,Keith Delman MD, Michael Ebright MD, Richard Wong MD, MaryseSt-Louis PhD*, Paul Johnson PhD*, Yuman Fong MD. MemorialSloan-Kettering Cancer Center, 564 First Avenue, Apartment 14C,New York, NY 10016, USA (212) 639-8421; *Neurovir Inc. Vancouver,Canada.

S68 Surgical Forum Abstracts J Am Coll Surg

Introduction: NV1034 is a novel multi-mutated, replication competentherpes simplex virus (HSV) type-I that combines two anticancer strat-egies: 1) Direct tumor oncolysis and 2) Immune modulation. It is anoncolytic virus that also carries the gene for murine GM-CSF. NV1034was compared to NV1020, the parent HSV mutant lacking GM-CSF.

Methods: Antitumor activity against CT26, the murine colorectal car-cinoma cell line was studied. Cytotoxicity and GM-CSF productionwere measured in cells infected in vitro with NV1034 or NV1020 atmultiplicity of infection (MOI) of 1. Efficacy of NV1034 and NV1020 wasthen tested in subcutaneous tumors in syngeneic Balb/c mice. Tumors(5–10 mm) were treated with 13107 plaque forming units of NV1020,NV1034 or PBS (controls) by single intratumoral (IT) injection (n 56–7/group). Tumor volumes were compared by Student’s t-test.

Results: In-vitro cytotoxicity assay demonstrated 96% and 82% tu-mor cell kill at 7 days after infection with NV1034 or NV1020 at an MOIof 1. ELISA demonstrated in-vitro (fig. 1) and in-vivo GM-CSF produc-tion in picogram quantities. Mean tumor volume (mm3) 6 SEM 2 weeksafter treatment was 710 6 203.3, 207.1 6 67.5 and 36.5 6 23 (p 5 0.01vs. control and 0.04 vs. IT NV1020) in the control, IT NV1020 and ITNV1034 groups (fig. 2).

Conclusions: NV1034 and NV1020 are cytotoxic to CT26 cells in-vitro.NV1034 has the ability to produce GM-CSF in-vitro and in-vivo. Effi-cacy of NV1034 for treating experimental colorectal carcinoma isgreater than that for NV1020 when delivered by IT injection. Combi-nation of oncolytic and immune therapy may be more effective intreating colorectal carcinoma.

Strong expression of sialyl-LEx defined by mAb AM-3IS, a prognostic marker after curative resection ofstage II and III colorectal carcinomasBenno Mann1, M.D., Patricia Grabowski2, M.D., Ulrich Mansmann3,Ph.D., Ernst-Otto Riecken2, M.D., Christoph Hanski2, Ph.D., Heinz-Johannes Buhr1, M.D. Departments of Surgery1, Gastroenterology2

and Medical Statistics3, UKBF, Freie Universität Berlin,Hindenburgdamm 30, 12200, Berlin, Germany Phone: 30 8445 2543,Fax: 30 8445 2740, e-mail: mann@ukbf.fu-berlin.de

Introduction: Sialyl-Lex antigen is overexpressed in 90% of colorectalcarcinomas. Binding of sialyl-Lex to E-selectin expressed on endothe-lial cells is supposed to be an initial step of metastatic tumor cellextravasation. The purpose of the present study was to analyze,whether sialyl-Lex expression increases with the progression of colo-rectal carcinoma stage and whether it has an independent prognosticimpact after surgical therapy.

Methods: 190 consecutive patients with complete 5-year follow-updata were analyzed. Sialyl-Lex was detected on paraffin-embeddedsections of the carcinomas by immunohistochemistry using mAbAM-3. A staining score resulting from staining frequency and intensitywas calculated and a cut-off point, separating patients with weak andstrong expression was determined using a matringale residual plot.The Jonckheere-Terpstra-test was used for the staining differences instage I–IV tumors. The predictive value of variables was analyzed withthe log-rank-test in univariate and with a Cox’s regression model inmultivariate analysis.

Results: The percentage of strongly expressing carcinomas increasedwith the progression of UICC stage (stage I 5 10%, II 5 48%, III 563% and IV 5 69%, p,0.0001). In multivariate analysis, strong sialyl-Lex expression showed to increase the relative risk of cancer relateddeath 3.8 fold (95%CI 1.8–7.9, see Table 1). The separate analyses ofall patients at stages II, III and IV revealed a significantly better 5-yearoverall-survival for patients with weakly expressing tumors after cura-

tive resection in stages II and III, but not in stage IV (stage II: 84 vs.55%, p 5 0.0013, III: 84 vs. 36%, p 5 0.00074, IV: 10 vs. 6%, p 50.27).

Table 1: Variables predicting overall-survival in 190 patients withcolorectal carcinoma

Univariate analysis Multivariate analysis

VariableChi-square p - value Variable r.r. 95% CI p - value

pT1-3 vs. pT4 11.5 5 0.0007 pT1-3 vs. T4 1.97 1.1–3.4 5 0.017pN0 vs. pN1 17.0 5 0.00004 pN0 vs. pN1 1.26 0.8–2.1 5 0.37M0 vs. M1 131.9 ,0.000001 M0 vs. M1 17.6 7.1–43.4 ,0.000001sLex 2 vs.1 9.0 5 0.0026 sLex 2 vs.1 3.81 1.8–7.9 5 0.00034

r.r. 5 relative risk; sLex 2 vs. 1 5 weak vs. strong immunohistochemical sialyl-Lex

expression; CI 5 confidence interval.

Conclusions: This data indicate that strong sialyl-LeX expression de-fined by mAb AM-3 is an independent unfavorable prognostic markerfor patients with colorectal carcinoma after curative resection in stagesII and III, whereas it does not influence the prognosis in patients whoalready developed distant metastases.

Human colon cancer cell growth is inhibited byadenoviral delivery of chimeric P27/P16Linda Martin, MD*, Sanjoy Dutta, MD*, Mary Matli, BS*, Salil Patel,PhD#, James McArthur, PhD#, Robert Warren, MD*. *UCSF, SanFrancisco, CA and #Cell Genesys, Foster City, CA. UCSF, Box 0790,533 Parnassus Ave., San Francisco, CA 94143-0790, USA. (415)476-0731

Introduction: Cyclin-dependent kinases play an important role in or-chestrating cell cycle progression. p16 and p27 are cyclin-dependentkinase inhibitors (CDKI) whose loss of function or down-regulation hasbeen implicated in tumor proliferation. Methylation of CpG islands onthe 59end of p16, leading to transcriptional inactivation, occurs fre-quently in colorectal tumors, and low levels of p27 are associated withpoor prognosis in colorectal cancer. Furthermore, reduced p27 ex-pression in metachronous colorectal metastases when compared toprimary tumors suggests that down-regulation of p27 may facilitatemetastatic spread. We have tested the ability of a chimeric p16/truncated p27 gene construct inserted in a replication-deficient adeno-viral vector to inhibit the growth of human colon cancer cells.

Methods: Two human colon cancer cell lines (HCT116 (p53 wt), DLD-1(mutant p53)) were chosen to test our hypothesis. Transduction effi-ciency was determined by flow cytometric analysis for each cell line 48hours after exposure to a replication-deficient adenoviral vector en-coding green fluorescent protein. Each cell line was then treated withreplication-deficient adenovirus encoding p27/p16 or p16, with mocktreatment and null adenovirus as controls. Cell number was deter-mined at the time of transduction (day 0) and 48 hours later. MTTassays were performed at days 0, 2, 4 and 6 to assess proliferation.Proliferative index and apoptosis were assessed by BrDU incorpora-tion and by TUNEL staining of DNA fragments, respectively.

Results: At a multiplicity of infection of 30, transduction efficacy inHCT116 and DLD-1 was greater than 99%. At 48 hours, treatment withp27/p16-Ad led to reductions in cell number of 74% for HCT116 and55% for DLD-1. In comparison, treatment with null-Ad resulted in adecrease in cell number (HCT116 by 47%, DLD-1 by 1%). After 6 days,MTT assays demonstrated further reductions in cell number: 78% inHCT, 64% in DLD-1 (p , 0.0001 for both by ANOVA). The decrease incell number was attributable to both a reduction in proliferative index(HCT116 by 99%; DLD-1 by 97%) and an increase in apoptotic index(19-fold for HCT116 and . 5-fold for DLD-1 compared to null-Adexposure). p16-Ad exposure resulted in cell number decreases inter-mediate to null-Ad and p27/p16-Ad exposure in both cell lines.

Conclusions: Adenoviral delivery of the CDKI chimera p27/p16 dra-matically decreased cell number and proliferative index in two humancolon cancer cell lines. These effects seem to be independent of p53status. Our findings suggest a new approach for gene therapy ofcolorectal cancer.

S69Vol. 191, No. 4S, October 2000 Surgical Forum Abstracts