William R. Wrightson

Complications associated with sentinel lymph node biopsy for melanoma

AbstractBackground: Sentinel lymph node (SLN) biopsy has become widely accepted as a method of staging the regional lymph nodes for patients with melanoma. Although it is often stated that SLN biopsy is a minimally invasive procedure associated with few complications, a paucity of data exists to specifically determine the morbidity associated with this procedure. This analysis was performed to evaluate the morbidity associated with SLN biopsy compared with completion lymph node dissection (CLND). Methods: Patients were enrolled in the Sunbelt Melanoma Trial, a prospective multi-institutional study of SLN biopsy for melanoma. Patients underwent SLN biopsy and were prospectively followed up for the development of complications associated with this technique. Patients who had evidence of nodal metastasis in the SLN underwent CLND. Complications associated with SLN biopsy alone were compared with those associated with SLN biopsy plus CLND. Results: A total of 2120 patients were evaluated, with a median follow-up of 16 months. Overall, 96 (4.6%) of 2120 patients developed major or minor complications associated with SLN biopsy, whereas 103 (23.2%) of 444 patients experienced complications associated with SLN biopsy plus CLND. There were no deaths associated with either procedure. Conclusions: SLN biopsy alone is associated with significantly less morbidity compared with SLN biopsy plus CLND.

Analysis of minocycline by high-performance liquid chromatography in tissue and serum

A sensitive and rapid reversed-phase high-performance liquid chromatography assay can be used to accurately determine serum and tissue minocycline concentrations. Minocycline is a broad spectrum tetracycline derivative with many applications. Tissue and serum samples were obtained from guinea pigs that had received either topical or intravenous minocycline. Samples were extracted using a Sep-Pak C18 cartridge and were injected into a microBondapak C18 column with an isocratic methanol mobile phase. Samples were analyzed using UV detection and produced sharp peaks with a retention time of 2.5 min. The lower limit of detection was 100 ng and drug recovery was 61%. This method greatly facilitated the analysis of minocycline while allowing for sensitivity.

Suppository delivery of 5-fluorouracil in rectal cancer

AbstractBackground: Preoperative radiotherapy with concomitant intravenous 5-fluorouracil (5-FU-IV) is effective in shrinking locally advanced rectal cancers and facilitating subsequent surgery. Topical 5-FU application may enhance its radiosensitizing and cytotoxic effects. Suppository and intravenous 5-FU administration were compared with respect to myelosuppression and tissue concentrations. Methods: Rats received 120 mg/kg 5-FU-IV or via suppository (5-FU-S). White blood cell count, serum albumin, alkaline phosphatase, creatinine, and aspartate aminotransferase (AST) were determined before and serially after 5-FU administration. In a separate experiment, rats received 5-FU-S or 5-FU-IV as already described. Portal and systemic blood, rectal, iliac lymph node, liver, and lung tissue were harvested for high-performance liquid chromatography determination of 5-FU concentrations 30 min, 1, 3, 6, and 12 h after drug administration. Results: No toxicity was observed in 5-FU-S animals, whereas 63% of 5-FU-IV animals had diarrhea. Weight loss and myelosuppression occurred only in 5-FU-IV animals. Rectal drug concentrations were significantly higher in the 5-FU-S animals compared with 5-FU-IV animals, 0.5–6 h after drug administration. Blood, liver, and lung 5-FU concentrations with 5-FU-S were comparable to those with 5-FU-IV. Conclusions: 5-FU suppositories are associated with fewer systemic side effects and higher rectal 5-FU concentrations than with 5-FU-IV administration.

C-terminal deletion mutant p21(WAF1/CIP1) enhances E2F-1-mediated apoptosis in colon adenocarcinoma cells

The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F-1 (Ad-E2F-1) and a C-terminal deletion mutant of p21WAF1/cIP1 (Ad-p21-PCNA), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F-1 and p21-PCNA overexpression in response to adenovirus infection was evident by Western blot analysis. IC25 concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F-1 and p21-PCNA resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly(ADP-ribose) polymerase (PARP) cleavage and analysis of cell morphology also revealed that coinfection with both Ad-E2F-1 and Ad-p21-PCNA enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F-1 protein expression was markedly enhanced in the E2F-1/p21-PCNA adenovirus combination compared to Ad-E2F-1 infection alone. However, these same effects were not evident in cells coinfected with Ad-E2F-1 and an adenovirus expressing wild-type human p21WAF1/CIP1 (Ad-p21WT). The increase in E2F-1 expression with coexpression of E2F-1 and p21-PCNA was not a result of increased E2F-1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single-gene therapy with either the wild-type or mutant p21, whereas increased accumulation of cells in G2/M phase was demonstrated in the E2F-1–overexpressing cells. In the combined therapies, E2F-1/p21-PCNA treatment still resulted in G1 arrest, but E2F-1 was able to counteract the G1 arrest when coinfected with p21WT. These results provide further evidence of the importance of the p21:PCNA-binding domain in mediating the complex cell cycle interaction between E2F-1 and p21. Simultaneous intratumoral injection of Ad-E2F-1 and Ad-p21-PCNA dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT-29 colon cancer xenografts. When combined with Ad-p21-PCNA, E2F-1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls (P<.05). In conclusion, although simultaneous delivery of E2F-1 and p21-PCNA transgenes results in increased E2F-1 expression and enhanced apoptosis of both SW620 and HT-29 colon cancer cells in vitro, this combination was only effective in the treatment of SW620 metastatic colon cancer in vivo. This may represent a potentially useful combination gene therapy strategy for metastatic colon cancer.

Suppository administration of chemotherapeutic drugs with concomitant radiation for rectal cancer

PURPOSE: Preoperative radiation with combined chemotherapy is effective in shrinking advanced rectal cancer locally and facilitating subsequent surgery. Suppository delivery of 5-fluorouracil is associated with less toxicity and higher rectal tissue concentrations than intravenous administration. This prompted us to evaluate suppository and intravenous administration of 5-fluorouracil and mitomycin C with concomitant radiation to determine associated toxicity. METHODS: Rectal, liver, lymph node, and lung tissue and systemic and portal blood were collected serially from male Sprague Dawley rats to determine drug concentrations following suppository or intravenous delivery of 5-fluorouracil or mitomycin C. Thirty-six animals were randomly assigned to treatment groups and received 5-fluorouracil suppositories, mitomycin C suppositories, or an equivalent intravenous dose of 5-fluorouracil or mitomycin C 30 minutes before radiation therapy. Before and 3, 6, 10, and 15 days following this treatment, blood was collected, colonoscopy was performed, and rectal tissue was harvested for histologic examination. RESULTS: Mitomycin C suppository was significantly less toxic compared with intravenous delivery, and higher rectal tissue concentrations were observed from 10 to 30 minutes (P< 0.05). Compared with intravenous 5-fluorouracil administration and radiation, 5-fluorouracil suppository and radiation resulted in additive myelosuppression at day 6 (P<0.05) with rapid recovery. CONCLUSIONS: 5-Fluorouracil and mitomycin C suppository delivery combined with radiation causes less systemic toxicity and is more effective than intravenous administration.