Judith M. Connett

Impact of pneumoperitoneum on trocar site implantation of colon cancer in hamster model

BACKGROUND: Numerous anecdotal reports have documented the spread of colon cancer to trocar sites after laparoscopic-assisted colectomy. We hypothesized that the pneumoperitoneum of laparoscopy potentiated tumor adherence to trocar sites. PURPOSE: This study was designed to determine the affect of CO2pneumoperitoneum on the rate of tumor implantation at trocar sites. METHODS: Viable GW-39 human colon cancer cells were injected into the abdominal cavity of hamsters (2 × 106cells/hamster). A midline laparotomy, insertion of four 5-mm trocars, injection of viable cells into the mesentery of the cecum, and free peritoneal cavity was performed in two groups: one control group (41) who did not receive a pneumoperitoneum and a comparison group (50) who underwent pneumoperitoneum for ten minutes at an insufflation pressure of 10 mmHg. Animals were killed at six weeks, and hematoxylin and eosin-stained sections of trocar wounds, midline wound, small intestine, cecum, liver, and lung were examined by a veterinary pathologist, who was blinded to operation. RESULTS: Pneumoperitoneum increased tumor implantation in the cecal mesentery and the midline incision (P<0.05) but did not effect recurrence in the liver, lung, or jejunum. Trocar site implantation tripled with the addition of pneumoperitoneum (26vs.75 percent) (P<0.0001). CONCLUSION: Pneumoperitoneum increased implantation of free intra-abdominal cancer cells at wound sites on the abdominal wall or within the abdominal cavity in this animal model. The use of pneumoperitoneum during laparoscopy in patients with colon cancer should only be performed in a protocol setting to evaluate the effect of pneumoperitoneum on the treatment of cancer.

Magnetic resonance imaging using gadolinium labeled monoclonal antibody

Gadolinium was attached to antibodies and tested in vitro and in vivo for its effect on proton relaxation enhancement. Using the cyclic anhydride method, diethylenetriaminepentaacetic acid (DTPA) was attached to albumin, IgG and anti-CEA monoclonal antibody. Gadolinium (Gd) was then chelated to the protein complexes forming protein-DTPA-Gd complex. With this technique approximately 9 atoms of Gd could be attached to each albumin molecule, 4 to each IgG molecule and 1.5 to each monoclonal antibody molecule. The minimal in vitro concentration of Gd in the form of IgG-DTPA-Gd necessary to produce proton relaxation enhancement at 0.35 tesla was 10(-1) mM. An in vivo experiment using anticarcinoembryonic antigen (CEA) monoclonal antibody-DTPA-Gd in hamsters implanted with human colon carcinoma resulted in a tumor concentration of Gd of less than 10(-4) mM. No enhancement of the tumors was detected at that concentration. For monoclonal antibodies to function as selective MR contrast agents, substantial advances in technology must occur.

Effects of pneumoperitoneum on tumor implantation with decreasing tumor inoculum.

INTRODUCTION: The aim of this study was to determine the effect of pneumoperitoneum on the rate of trocar-site implantation with decreasing inoculum of cancer cells. METHODS: A total of 0.5 ml of GW-39 human colon cancer cell suspensions at 1 percent (∼3.2×105 cells) and at 0.5 percent (∼1.6×105 cells; v/v) were injected into the abdomen of hamsters through a midline incision. Animals in each group were randomized to receive either pneumoperitoneum (1 percent=33; 0.5 percent=43) or not (1 percent=32; 0.5 percent=39). Gross and microscopic tumor implants were documented seven weeks later at four trocar sites. RESULTS: In the 1 percent group, pneumoperitoneum significantly increased trocar-site tumor implants from 50 to 71 percent (P<0.001). Pneumoperitoneum also resulted in the following: 1) more frequent involvement of all four concurrent sites (38vs. 10 percent;P<0.02); 2) more frequent palpable tumors (13vs. 5 percent;P<0.01); 3) larger tumor mass (2.1±0.6 gvs. 0.2±0.1 g;P<0.02). In the 0.5 percent group, pneumoperitoneum did not significantly increase trocar-site tumor implants, and it did not result in a larger tumor mass. The percent increase in trocar-site implants owing to pneumoperitoneum was influenced by the amount of tumor inoculum (21 percent in the 1 percent group; 10 percent in the 0.5 percent group). The mass of palpable tumor implants after pneumoperitoneum decreased with decreased inoculum: 1 percent =2.1±0.6 g; 0.5 percent=0.3±0.1 g (P<0.0001). CONCLUSIONS: Pneumoperitoneum significantly increased both tumor implantation rate and mass when ∼3.2×105 colon cancer cells were injected into the peritoneal cavity. These effects of pneumoperitoneum diminished with one-half as many tumor cells injected in the peritoneal cavity.

Excision of trocar sites reduces tumor implantation in an animal model

PURPOSE: The purpose of this study was to determine the effect of excising abdominal trocar wound sites after pneumoperitoneum on the rate of trocar site tumor implantation in a hamster model. This would help determine whether tumor cells seed trocar sites during or after pneumoperitoneum. METHODS: A total of 0.5 ml of GW-39 human colon cancer cell suspension at 2.5 percent v/v (8×105 cells) was injected into the abdomens of 77 hamsters through a midline incision. Animals were subjected to ten minutes of pneumoperitoneum, after placement of four abdominal trocars, and then randomly assigned to undergo either simple suture closure or 4-mm radius trocar wound site excision at the end of the procedure. Gross and microscopic tumor implants were documented seven weeks later. RESULTS: There were three and four deaths in simple suture closure and wound site excision groups, respectively. Of the remaining 35 hamsters in each group, tumor cells implanted at 89 and 78 percent of trocar sites, respectively (P<0.03). There was no significant difference between the two groups in tumor implantation at midline laparotomy sites. Wound site excision also resulted in fewer palpable tumors (44vs. 61 percent;P<0.01) and a lower tumor implantation rate (49vs. 74 percent;P<0.05) at all four concurrent sites compared with simple suture closure. CONCLUSIONS: Excision of laparoscopic abdominal trocar wound sites can significantly, but not completely, reduce tumor implantation rate compared with simple wound closure.

Conjugation of Monoclonal Antibodies with TETA Using Activated Esters: Biological Comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3

A simple method for conjugation of monoclonal antibodies (mAbs) with the chelating agent 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been developed using commercially available reagents. This method involved activation of a single carboxyl group of TETA with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The resulting activated ester of TETA was reacted with the anti-colorectal carcinoma mAb 1A3 at molar ratios ranging from 10:1 to 100:1 to give immunoconjugates modified with an average of 0.4 to 2.0 functional chelators per antibody molecule. The TETA-1A3 conjugate was labeled with 64Cu at specific activities as high as 15.4 microCi/microgram, and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity. The biodistribution of 64Cu-labeled TETA-1A3 in hamsters bearing GW39 human colon carcinoma xenografts was compared to that of 64Cu-BAT-2IT-1A3 (BAT = 6-(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid; 2IT = 2-iminothiolane). Both conjugates showed high tumor uptake (6.60-9.05% injected dose/gram) from 24 to 48 h post-injection and generally similar blood clearance and non-target organ uptakes. Human absorbed dose estimates derived from the hamster biodistribution data showed the critical organs for both conjugates to be the large intestine and the red marrow. Our results suggest that the in vitro and in vivo performance characteristics of 64Cu-TETA-1A3 compare favorably with those of 64Cu-BAT-2IT-1A3 and that further evaluation of the diagnostic and therapeutic efficacy of 64Cu-TETA-1A3 is warranted.

Investigation of copper-PTSM as a PET tracer for tumor blood flow

Copper-PTSM has been shown in previous studies to act as a fluid microsphere and to be useful in quantitating blood flow in brain, myocardium, and kidneys. In this study we have evaluated this agent as a PET tumor blood flow agent. 64Cu- or 67Cu-labeled Cu-PTSM was administered (i.v.) to Golden Syrian hamsters with colorectal carcinoma cell implants (GW39). One minute prior to sacrifice (10-60 min after Cu-PTSM was administered) 125I-iodoantipyrine (125I-IAP), an agent known to measure tumor blood flow, was administered intravenously by a 3-stage, 1 min ramp infusion. Following sacrifice, samples of tumor and brain were removed (within 40s) and the tumor and brain levels of Cu-PTSM and iodoantipyrine determined. Since the brain uptake of both Cu-PTSM and IAP is perfusion rate limited, the brain was used as a reference organ to normalize tumor levels of the two tracers. The plot of Cu-PTSM versus 125I-IAP tumor/brain ratios showed a good linear correlation (r value of 0.97), suggesting that Cu-PTSM could be used to quantify tumor blood flow. Since the mechanism of Cu-PTSM trapping is likely to be due to glutathione levels in the tissue, and because tumor tissue glutathione levels might vary, the temporal uptake of Cu-PTSM was investigated by PET imaging both the tumor-bearing hamsters and approximately 300 g Copenhagen rats bearing R3227 prostate tumors. The tumors were clearly visualized and the retained copper radioactivity in the tumor was constant over the 30 min imaging period.

Local treatment of abdominal wound reduces tumor implantation

Background and Objectives: Pneumoperitoneum increases the trocar‐site tumor implantation rate using a human colon cancer cell line in a hamster model. The purpose of this study was to determine whether local treatment of trocar sites with potential tumoricidal agents can inhibit tumor implantation after pneumoperitoneum.

Sodium hyaluronate carboxymethylcellulose-based bioresorbable membrane (seprafilm™)—Does it affect tumor implantation at abdominal wound sites?

PURPOSE: This study examines the effects of a sodium hyaluronate-based bioresorbable membrane (Seprafilm™) on tumor implantation at surgical wound and laparoscopic trocar sites. METHODS: GW-39, an established human colon cancer line carried in immunocompetent golden Syrian hamsters was used as the experimental model. Under general anesthesia, a 2-cm midline incision was made to allow placement of four 5-mm abdominal trocars. Hamsters were then randomly assigned to preSeprafilm™, postSeprafilm™, and control (no Seprafilm™) groups. In the preSeprafilm™ group 0.5 ml of a 5 percent (vol/vol) suspension of the GW-39 tumor cells (∼1.675 × 106 cells) was injected into the abdomen of each hamstervia midline incision. Trocars were removed, the wounds were closed, and 1 cm2 of Seprafilm™ was placed on the peritoneal surface of each trocar site. In the postSeprafilm™ group the membrane was placed at each site before injection of tumor cells. The control group did not receive Seprafilm™. The animals were killed after seven weeks, and the abdominal wound sites were excised. Sites without gross tumor underwent histologic evaluation. RESULTS: One hundred thirty-two animals were randomly assigned to the three groups. The preSeprafilm™ group had an 87 percent tumor implantation rate. The postSeprafilm™ group had a 90 percent tumor implantation rate. The control group had an 88 percent tumor implantation rate. Chi squared analysis demonstrated that these total tumor implant rates and mean tumor mass were similar at all wound sites and between groups. No toxicity was observed in any of the experimental groups. CONCLUSIONS: Sodium hyaluronate-based bioresorbable membrane (Seprafilm™) does not influence GW-39 human colon cancer implantation at abdominal wound sites in this hamster model.

Pneumoperitoneum does not influence trocar site implantation during tumor manipulation in a solid tumor model.

BackgroundThe purpose of this study was to assess tumor implantation at abdominal wound sites following manipulation of a solid abdominal tumor.MethodsGW-39 human colon cancer cells were injected into the omentum of golden Syrian hamsters. At 2 weeks, an omental tumor was harvested and animals were randomized to bivalve (A), crush (B), strip (C), or excision (D), with or without pneumoperitoneum. Four 5-mm trocars were inserted into the abdomen, and the tumor was reinserted through the midline, swept through four quadrants, and removed. The incision was closed and pneumoperitoneum at 7 mmHg was maintained for 10 min. Tumor implantation at wound sites was documented at 7 weeks.ResultsImplantation at trocar sites was 53 and 49% with and without pneumoperitoneum in the manipulated groups (A, B, C), respectively (p = 0.993). Implantation at trocar sites was reduced in the control group (D) at 9 and 10% with and without pneumoperitoneum, respectively (p < 0.001).ConclusionsTumor implantation at trocar sites is due to spillage of tumor during manipulation and not to pneumoperitoneum.

Non-histone chromosomal proteins from immunoglobulin-producing mouse plasmacytoma cells.

Abstract Non-histone chromosomal (MHC proteins from two immunoglobulin-producing mouse plasmacytoma cell lines, from three variant lines derived from these parentals, and from control mouse lung fibroblast L929 cells have been examined using two-dimensional polyacrylamide gel electrophoresis. When NHC protein profiles of the plasmacytoma lines were compared, three proteins were identified whose presence correlated with the synthesis of the immunoglobulin γ 2b heavy chain. However, the overall NHC protein patterns of the different plasmacytoma lines were very similar: an average of 95% of the proteins in one line were shared with the others. In contrast, fewer than 50% of mouse plasmacytoma NHC proteins were identified in mouse L929 protein profiles. Surprisingly, proteins with the electrophoretic and serological properties of immunoglobulin chains were also detected in the nucleoplasm and in the NHC protein populations of lines synthesizing these proteins. A control preparation indicated that these proteins were probably not the result of random contamination of NHC proteins by cytoplasmic immunoglobulin chains.