Safety and advantages of combined resection and microwave ablation in patients with bilobar hepatic malignancies.

BACKGROUND: The multimodality approach has significantly improved outcomes for hepatic malignancies. Microwave ablation is often used in isolation or succession, and seldom in combination with resection. Potential benefits and pitfalls from combined resection and ablation therapy in patients with complex and extensive bilobar hepatic disease have not been well defined. METHODS: A review of the University of Louisville prospective Hepato-Pancreatico-Biliary Patients database was performed with multi-focal bilobar disease that underwent microwave ablation with resection or microwave only included. RESULTS: One hundred and eight were treated with microwave only (MWA, n = 108) or combined resection and ablation (CRA, n = 84) and were compared with similar disease-burden patients undergoing resection only (n = 84). The groups were comparable except that the MWA group was older (p = .02) and with higher co-morbidities (diabetes, hepatitis). The resection group had larger tumours (4 vs. 3.2 and 3 cm) but the CRA group had more numerous lesions (4 vs. 3 and 2, p = .002). Short-term outcomes including morbidity (47.6% vs. 43%, p = .0715) were similar between the CRA and resection only groups. Longer operative time (164 vs. 126 min, p = .003) and need for blood transfusion (p = .001) were independent predictors of complications. Survival analyses for colorectal metastasis patients (n = 158) demonstrated better overall survival (OS) (43.9 vs. 37.6 and 30.5 months, p = .035), disease-free survival (DFS) (38 vs. 26.6 and 16.9 months, p = .028) and local recurrence-free survival (LRFS) (55.4 vs. 17 and 22.9 months, p < .001) with resection only. CONCLUSION: The use of microwave ablation in addition to surgical resection did not significantly increase the morbidities or short-term outcomes. In combination with systemic and other local forms of therapy, combined resection and ablation is a safe and effective procedure.

Efficacy of preoperative immunonutrition in locally advanced pancreatic cancer undergoing irreversible electroporation (IRE).

BACKGROUND: Improved preoperative immunonutrition has been shown to decrease the length of stay (LOS) and complications among patients undergoing elective gastrointestinal cancer surgeries. The purpose of this study was to determine whether preoperative immunonutrition supplementation decreases postoperative LOS, infectious complications, and morbidity in patients undergoing irreversible electroporation (IRE) surgery for locally advanced pancreatic cancer (LAPC). METHODS: At a regional hepatopancreatobiliary referral center within an academic medical center 71 patients receiving IRE treatment of LAPC were included in the study. The participants were divided into those receiving preoperative immunonutrition (n = 44) and those receiving no supplemental preoperative immunonutrition (n = 27). Main outcomes and measures were LOS, postoperative complications, nutritional risk index (NRI), and albumin levels. RESULTS: Patients in both groups were similar for preoperative nutrition parameters and operative therapy. Patients in the immunonutrition group experienced a statistically significant decrease in postoperative complications (p = 0.05) and LOS (10.7 vs. 17.4, p = 0.01), and less of a decrease in nutritional risk index (-12.6 vs. -16.2, p = 0.03) and albumin levels (-1.1 vs. -1.5, p < 0.01). CONCLUSION: Preoperative immunonutrition was clinically significant in decreasing postoperative complications, LOS, and improving post-surgery NRI and albumin levels in patients receiving elective IRE treatment of non-resectable pancreatic cancer. These results indicate that preoperative immunonutrition is effective and feasible in this subset of cancer patients.

Selective replication of E1B55K-deleted adenoviruses depends on enhanced E1A expression in cancer cells.

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

Intraoperative magnetic resonance imaging for ablation of hepatic tumors.

BACKGROUND: The most significant rise in the use of hepatic ablation has come from image-guided techniques with both computed tomography (CT) and ultrasound (US). The recent development of open-configuration magnetic resonance scanners has opened up an entire new area of image-guided surgical and interventional procedures. Thus the aim of this study was to evaluate the use of intraoperative MRI (iMRI) ablation of hepatic tumors performed by surgeons. METHOD: Percutaneous iMRI hepatic ablation was performed from January 2003 to February 2005 for control of either primary or secondary hepatic disease. RESULTS: Eighteen hepatic ablations were performed on 11 patients with a median age of 71 (range: 51-81) years for metastatic colorectal cancer (n = 6), hepatocellular cancer (n = 2), cholangiocarcinoma (n = 2), and metastatic neuroendocrine (n = 1). Median hospital stay was 1 day, with complications occurring in 2 patients. After a median follow up of 18 months, there have been no local ablation recurrences, 5 patients are free of disease, 4 are alive with disease, 1 has died of disease, and 1 has died of other causes. CONCLUSIONS: Image-guided hepatic ablations represent a useful technique in managing hepatic tumors. Intraoperative MRI represents a new technique with initial success that has been limited to European centers. Further evaluation in U.S. centers has demonstrated iMRI to be useful for certain hepatic tumors that cannot be adequately visualized by US or CT.

mdm2 deletion does not alter growth characteristics of p53-deficient embryo fibroblasts.

The mdm2 gene encodes a protein that is necessary for the negative regulation of p53 function in vivo. Deletion of the mdm2 gene in mice results in early embryonic death while concomitant mdm2 and p53 deletion results in viable offspring. The viability of these mice prompted us to ask if MDM2 had an important growth regulatory function independent of p53. We established mouse embryo fibroblasts null for both p53 and mdm2 and compared them with p53-null fibroblasts. The cells did not differ in their growth rates or their ability to bypass a G1 arrest. Both cell lines formed colonies efficiently when plated at low density and showed a similar degree of genetic instability. Thus, the analysis of several growth parameters indicated no difference between p53-null and p53/mdm2-null cell lines.

Sentinel lymph node biopsy for melanoma: controversy despite widespread agreement.

Although sentinel lymph node (SLN) biopsy for melanoma has been adopted throughout the United States and abroad as a standard method of determining the pathologic status of the regional lymph nodes, some controversy still exists regarding the validity and utility of this procedure. SLN biopsy is a minimally invasive procedure, performed on an outpatient basis at the time of wide local excision of the melanoma, with little morbidity. Numerous studies have documented the accuracy of this procedure for identifying nodal metastases. There are four major reasons to perform SLN biopsy. First, SLN biopsy improves the accuracy of staging and provides valuable prognostic information for patients and physicians to guide subsequent treatment decisions. Second, SLN biopsy facilitates early therapeutic lymph node dissection for those patients with nodal metastases. Third, SLN biopsy identifies patients who are candidates for adjuvant therapy with interferon alfa-2b. Fourth, SLN biopsy identifies homogeneous patient populations for entry onto clinical trials of novel adjuvant therapy agents. Overall, the benefit of accurate nodal staging obtained by SLN biopsy far outweighs the risks and has important implications for patient management.

Sentinel lymph node biopsy for breast cancer: a suitable alternative to routine axillary dissection in multi-institutional practice when optimal technique is used.

PURPOSE: Previous studies have demonstrated the feasibility of sentinel lymph node (SLN) biopsy for nodal staging of patients with breast cancer. However, unacceptably high false-negative rates have been reported in several studies, raising doubt about the applicability of this technique in widespread surgical practice. Controversy persists regarding the optimal technique for correctly identifying the SLN. Some investigators advocate SLN biopsy using injection of a vital blue dye, others recommend radioactive colloid, and still others recommend the use of both agents together. PATIENTS AND METHODS: A total of 806 patients were enrolled by 99 surgeons. SLN biopsy was performed by single-agent (blue dye alone or radioactive colloid alone) or dual-agent injection at the discretion of the operating surgeon. All patients underwent attempted SLN biopsy followed by completion level I/II axillary lymph node dissection to determine the false-negative rate. RESULTS: There was no significant difference (86% v 90%) in the SLN identification rate among patients who underwent single- versus dual-agent injection. The false-negative rates were 11.8% and 5.8% for single- versus dual-agent injection, respectively (P <.05). Dual-agent injection resulted in a greater mean number of SLNs identified per patient (2. 1 v 1.5; P <.0001). The SLN identification rate was significantly less for patients older than 50 years as compared with that of younger patients (87.6% v 92.6%; P =.03). Upper-outer quadrant tumor location was associated with an increased likelihood of a false-negative result compared with all other locations (11.2% v 3. 9%; P <.05). CONCLUSION: In multi-institutional practice, SLN biopsy using dual-agent injection provides optimal sensitivity for detection of nodal metastases. The acceptable SLN identification and false-negative rates associated with the dual-agent injection technique indicate that this procedure is a suitable alternative to routine axillary dissection across a wide spectrum of surgical practice and hospital environments.

Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system.

PURPOSE: The American Joint Committee on Cancer (AJCC) recently proposed major revisions of the tumor-node-metastases (TNM) categories and stage groupings for cutaneous melanoma. Thirteen cancer centers and cancer cooperative groups contributed staging and survival data from a total of 30,450 melanoma patients from their databases in order to validate this staging proposal. PATIENTS AND METHODS: There were 17,600 melanoma patients with complete clinical, pathologic, and follow-up information. Factors predicting melanoma-specific survival rates were analyzed using the Cox proportional hazards regression model. Follow-up survival data for 5 years or longer were available for 73% of the patients. RESULTS: This analysis demonstrated that (1) in the T category, tumor thickness and ulceration were the most powerful predictors of survival, and the level of invasion had a significant impact only within the subgroup of thin (< or = 1 mm) melanomas; (2) in the N category, the following three independent factors were identified: the number of metastatic nodes, whether nodal metastases were clinically occult or clinically apparent, and the presence or absence of primary tumor ulceration; and (3) in the M category, nonvisceral metastases was associated with a better survival compared with visceral metastases. A marked diversity in the natural history of pathologic stage III melanoma was demonstrated by five-fold differences in 5-year survival rates for defined subgroups. This analysis also demonstrated that large and complex data sets could be used effectively to examine prognosis and survival outcome in melanoma patients. CONCLUSION: The results of this evidence-based methodology were incorporated into the AJCC melanoma staging as described in the companion publication.

Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.

PURPOSE: To revise the staging system for cutaneous melanoma under the auspices of the American Joint Committee on Cancer (AJCC). MATERIALS AND METHODS: The prognostic factors analysis described in the companion publication (this issue), as well as evidence from the published literature, was used to assemble the tumor-node-metastasis criteria and stage grouping for the melanoma staging system. RESULTS: Major changes include (1) melanoma thickness and ulceration but not level of invasion to be used in the T category (except for T1 melanomas); (2) the number of metastatic lymph nodes rather than their gross dimensions and the delineation of clinically occult (ie, microscopic) versus clinically apparent (ie, macroscopic) nodal metastases to be used in the N category; (3) the site of distant metastases and the presence of elevated serum lactic dehydrogenase to be used in the M category; (4) an upstaging of all patients with stage I, II, and III disease when a primary melanoma is ulcerated; (5) a merging of satellite metastases around a primary melanoma and in-transit metastases into a single staging entity that is grouped into stage III disease; and (6) a new convention for defining clinical and pathologic staging so as to take into account the staging information gained from intraoperative lymphatic mapping and sentinel node biopsy. CONCLUSION: This revision will become official with publication of the sixth edition of the AJCC Cancer Staging Manual in the year 2002.

E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells.

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.

Caspase activation and changes in Bcl-2 family member protein expression associated with E2F-1-mediated apoptosis in human esophageal cancer cells.

The prognosis for patients with esophageal cancer remains poor, prompting the search for new treatment strategies. Overexpression of E2F-1 has been shown to induce apoptosis in several cancer cell types. In the present study, the effect of adenovirus-mediated E2F-1 overexpression on human esophageal cancer cell lines Yes-4 and Yes-6 was evaluated. Cells were treated by mock infection, infection with an adenoviral vector expressing beta-galactosidase (Ad5CMV-LacZ), or E2F-1 (Ad5CMVE2F-1). Western blot analysis confirmed marked overexpression of E2F-1 in Ad5CMVE2F-1-infected cells. Overexpression of E2F-1 resulted in marked growth inhibition and rapid loss of cell viability due to apoptosis, although Yes-6 cells were somewhat more resistant to E2F-1-mediated growth inhibition than Yes-4 cells. Cell cycle analysis revealed that overexpression of E2F-1 led to G2 arrest, followed by apoptotic cell death. p53 expression remained undetectable in both cell lines after E2F-1 overexpression. The apoptosis inhibitor proteins of the Bcl-2 gene family, Bcl-2, Mcl-1, and BcI-XL, decreased at 48 h after infection in Yes-4 cells, but remained unchanged in Yes-6 cells. Levels of retinoblastoma gene product (pRb) declined at 48 h after E2F-1 infection in Yes-4 cells, at which apoptosis predominated, whereas pRb expression remained constant in Yes-6 cells. Expression of p14ARF did not change after E2F-1 infection in either cell line. Involvement of caspase 3 and caspase 6 in E2F-1-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and poly-ADP-ribose polymerase, as well as fragmentation of the caspase 6 substrate, lamin B. These results indicate that the sensitivity of esophageal cancer cells to E2F-1-mediated apoptosis may be related to differential expression of Bcl-2 family member proteins and suggest that the adenovirus-mediated E2F-1 gene therapy may be a promising treatment strategy for the treatment of this disease.

Adenovirus-mediated E2F-1 gene transfer inhibits MDM2 expression and efficiently induces apoptosis in MDM2-overexpressing tumor cells.

The oncoprotein MDM2 binds and inactivates p53. MDM2 also binds to the tumor suppressor pRB, as well as E2F-1. E2F-1 is a transcription factor that regulates S phase entry and has been shown to cause apoptosis in some cell types when overexpressed. To investigate the effect of adenovirus-mediated E2F-1 overexpression, MDM2-overexpressing tumor cell lines were treated by mock infection, infection with an adenoviral vector expressing beta galactosidase, or E2F-1 (Ad5CMV-E2F-1). Western blot analysis confirmed significant overexpression of E2F-1 in Ad5CMV-E2F-1-infected cells. E2F-1 overexpression resulted in marked growth inhibition and rapid loss of cell viability. Ad5CMV-E2F-1 infection resulted in early S phase entry, followed by apoptotic cell death. E2F-1 overexpression was associated with a marked decrease in MDM2 levels and no evidence of increased Bax levels, whereas p53 and Bcl-2 levels remained undetectable. Cleavage of poly-ADP-ribose polymerase and caspase 3/CPP32 implicated activation of the caspase cascade in E2F-1-mediated apoptosis. These results indicate that adenovirus-mediated E2F-1 overexpression in MDM2-overexpressing tumor cells results in decreased MDM2 expression and widespread apoptosis. Because MDM2-overexpressing tumors are often resistant to p53 gene therapy, adenovirus-mediated E2F-1 gene therapy may be a promising alternative strategy.

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells.

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.

Additive effect of adenovirus-mediated E2F-1 gene transfer and topoisomerase II inhibitors on apoptosis in human osteosarcoma cells.

Recently, it has been demonstrated that Etoposide, a topoisomerase II inhibitor, can induce apoptosis in MDM2-overexpressing tumor cells by inhibition of MDM2 synthesis. We have previously shown that E2F-1 overexpression induces apoptosis of MDM2-overexpressing sarcoma cells, which is related to the inhibition of MDM2 expression. Therefore, the present study was designed to investigate the in vitro and in vivo effect of combined treatment of adenovirus-mediated E2F-1 and topoisomerase II inhibitors on the growth inhibition and apoptosis in human sarcoma cells. Two human sarcoma cell lines, OsACL and U2OS, were treated with topoisomerase II inhibitors (Etoposide and Adriamycin), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1). E2F-1 expression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at a low dose (multiplicity of infection, 2) markedly increased the sensitivity of human sarcoma cells to topoisomerase II inhibitor treatment. This cooperative effect of E2F-1 and topoisomerase II inhibitors was less marked in SAOS-2 cells (p53 and pRb null). Topoisomerase II inhibitors also cooperated with E2F-1 overexpression to enhance tumor cell killing in an in vivo model using xenografts in nude mice. When combined with Adriamycin or Etoposide, E2F-1 adenovirus therapy resulted in approximately 95% and 85% decrease in tumor size, respectively, compared to controls (P<.05). These results suggest a new chemosensitization strategy that is effective in MDM2-overexpressing tumors and may have clinical utility.

Passive immunization against tumor necrosis factor and interleukin-1 fails to reduce lung neutrophil sequestration in chronic sepsis.

The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are produced within the lung during sepsis, and may induce neutrophil sequestration resulting in neutrophil-mediated lung injury. We hypothesized that, if there is a cause and effect between TNF alpha or IL-1 production and lung neutrophil sequestration during chronic sepsis, TNF alpha mRNA and IL-1 mRNA levels in the lung after cecal ligation and puncture should correlate with the number of sequestered neutrophils as measured by the myeloperoxidase (MPO) content of the lung. To test this hypothesis, Swiss Webster mice were subjected to varying degrees of infectious challenge by single and double-puncture cecal ligation and puncture, or simultaneous antibiotic treatment, and their lungs and blood were harvested at 24 h. Lung TNF alpha and IL-1 beta mRNAs were measured by the reverse-transcription differential polymerase chain reaction, and MPO was measured by colorimetric assay. TNF alpha serum levels showed no correlation with the MPO content of the lung, whereas IL-1 levels were undetectable. Lung TNF alpha mRNA correlated weakly, and IL-1 beta mRNA exhibited a strong correlation with lung MPO (r = .9, p < .01), but administration of anti-TNF alpha- or anti-IL-1-neutralizing antibodies did not prevent a rise in lung MPO. IL-1 beta mRNA in bronchoalveolar macrophages correlated well with whole lung tissue IL-1 beta mRNA levels (r = .91, p < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of mononuclear cell immune-associated antigen expression, interleukin-1 expression, and antigen presentation during intra-abdominal infection.

Intact peritoneal macrophage (M phi) function is critical to successful localization of intra-abdominal infection. Peritoneal macrophage antigen presentation capacity (APC), interleukin-1 (IL-1) expression, and immune-associated (Ia) antigen expression and abscess formation were determined following cecal ligation and puncture. APC and IL-1 expression were measured by coculture with a T-helper cell clone and by measuring subsequent proliferation. Ia expression was determined in blood, peritoneal M phi, and splenocytes using anti-Ia monoclonal antibody stain and flow cytometric analysis. Significant reductions in both Ia expression and APC were found 1 and 4 days after CLP with no change in IL-1 expression. Muramyl dipeptide, which enhances M phi phagocytosis, partially abrogated the depression in antigen presentation but did not affect Ia expression. Peritoneal M phi Ia expression and APC, but not IL-1 expression, were depressed after experimental peritonitis. The recovery of M phi function by day 14 coincides with clinical recovery and abscess formation, and restoration of early M phi depression may improve outcome.

p53 gene transfer does not enhance E2F-1-mediated apoptosis in human colon cancer cells.

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.

Enhanced survival from cecal ligation and puncture with pentoxifylline is associated with altered neutrophil trafficking and reduced interleukin-1 beta expression but not inhibition of tumor necrosis factor synthesis.

BACKGROUND: Our preliminary results showed that pentoxifylline improves survival after cecal ligation and puncture (CLP), even though in this model inhibition of tumor necrosis factor (TNF) activity decreases survival. In this study we tested the hypothesis that pentoxifylline improves survival after CLP, not by inhibiting TNF synthesis but by exerting its effect on leukocyte adhesiveness, neutrophil sequestration, recruitment of cells into the focus of sepsis, and interleukin-1 (IL-1) expression. METHODS: Pentoxifylline, 10 or 100 mg/kg/day, was administered to mice after CLP by infusion for 3 days. The following was measured at 24 hours for the group with improved survival: (1) serum TNF by enzyme-linked immunosorbent assay, (2) TNF and IL-1 beta mRNAs in lung and peritoneal macrophages by the differential polymerase chain reaction, (3) lung myeloperoxidase by a colorimetric assay, (4) leukocyte CD11b/CD18 by flow cytometry, and (5) peritoneal exudate cells by manual counting. RESULTS: Only the low-dose pentoxifylline increased survival. Pentoxifylline reduced IL-1 beta mRNA expression in lung and peritoneal macrophages but not TNF mRNA or immunoreactive TNF in the serum. The myeloperoxidase content of lung was reduced by pentoxifylline, but leukocyte CD11b/CD18 expression did not change. Pentoxifylline increased the number of cells in the peritoneum after CLP. CONCLUSIONS: Pentoxifylline improves survival after CLP without inhibiting TNF synthesis or expression of CD11b/CD18 on leukocytes. Pentoxifylline treatment reduced lung neutrophil sequestration and IL-1 beta mRNA levels and increased cell recruitment in the peritoneum.

Factors that predict the presence of sentinel lymph node metastasis in patients with melanoma.

BACKGROUND: This analysis was performed to identify prognostic factors that are predictive of sentinel lymph node (SLN) metastasis in melanoma. METHODS: Analysis was performed of a multi-institutional, prospective, randomized trial of SLN biopsy for melanoma. Eligibility criteria included age 18 to 70 years, Breslow thickness of 1.0 mm or more, and clinically negative regional lymph nodes. SLNs were evaluated by serial sectioning and immunohistochemistry for S100. Univariate chi-square and multivariate logistic regression analyses were performed to assess factors predictive of the presence of a positive SLN. Probability values of less than.05 were considered significant. RESULTS: SLNs were identified in 99.7% of patients. A total of 1058 patients were evaluated; 961 patients had complete data and were included in the statistical analysis. SLNs were positive for tumor in 208 of 961 patients (22%). Breslow thickness, Clark level, ulceration, and patient age were factors that were found to be independently predictive of the presence of SLN metastasis. CONCLUSIONS: Increasing Breslow thickness, Clark level of more than III, the presence of ulceration, and patient age of 60 years or less are the most important independent prognostic factors associated with the finding of positive SLN in patients with melanoma.

Practical guidelines for optimal gamma probe detection of sentinel lymph nodes in breast cancer: results of a multi-institutional study. For the University of Louisville Breast Cancer Study Group.

INTRODUCTION: Multiple radioactive lymph nodes are often removed during the course of sentinel lymph node (SLN) biopsy for breast cancer when both blue dye and radioactive colloid injection are used. Some of the less radioactive lymph nodes are second echelon nodes, not true SLNs. The purpose of this analysis was to determine whether harvesting these less radioactive nodes, in addition to the "hottest" SLNs, reduces the false-negative rate. METHODS: Patients were enrolled in this multicenter (121 surgeons) prospective, institutional review board-approved study after informed consent was obtained. Patients with clinical stage T1-2, N0, M0 invasive breast cancer were eligible. This analysis includes all patients who underwent axillary SLN biopsy with the use of an injection of both isosulfan blue dye and radioactive colloid. The protocol specified that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest node should be removed and designated SLNs. All patients underwent completion level I/II axillary dissection. RESULTS: SLNs were identified in 672 of 758 patients (89%). Of the patients with SLNs identified, 403 patients (60%) had more than 1 SLN removed (mean, 1.96 SLN/patient) and 207 patients (31%) had nodal metastases. The use of filtered or unfiltered technetium sulfur colloid had no impact on the number of SLNs identified. Overall, 33% of histologically positive SLNs had no evidence of blue dye staining. Of those patients with multiple SLNs removed, histologically positive SLNs were found in 130 patients. In 15 of these 130 patients (11.5%), the hottest SLN was negative when a less radioactive node was positive for tumor. If only the hottest node had been removed, the false-negative rate would have been 13.0% versus 5.8% when all nodes with 10% or more of the ex vivo count of the hottest node were removed (P =.01). CONCLUSIONS: These data support the policy that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest SLN should be harvested for optimal nodal staging.