R. Y. Declan Fleming

Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle.

We have investigated the mechanisms of the anabolic effect of insulin on muscle protein metabolism in healthy volunteers, using stable isotopic tracers of amino acids. Calculations of muscle protein synthesis, breakdown, and amino acid transport were based on data obtained with the leg arteriovenous catheterization and muscle biopsy. Insulin was infused (0.15 mU/min per 100 ml leg) into the femoral artery to increase femoral venous insulin concentration (from 10 +/- 2 to 77 +/- 9 microU/ml) with minimal systemic perturbations. Tissue concentrations of free essential amino acids decreased (P < 0.05) after insulin. The fractional synthesis rate of muscle protein (precursor-product approach) increased (P < 0.01) after insulin from 0.0401 +/- 0.0072 to 0.0677 +/- 0.0101%/h. Consistent with this observation, rates of utilization for protein synthesis of intracellular phenylalanine and lysine (arteriovenous balance approach) also increased from 40 +/- 8 to 59 +/- 8 (P < 0.05) and from 219 +/- 21 to 298 +/- 37 (P < 0.08) nmol/min per 100 ml leg, respectively. Release from protein breakdown of phenylalanine, leucine, and lysine was not significantly modified by insulin. Local hyperinsulinemia increased (P < 0.05) the rates of inward transport of leucine, lysine, and alanine, from 164 +/- 22 to 200 +/- 25, from 126 +/- 11 to 221 +/- 30, and from 403 +/- 64 to 595 +/- 106 nmol/min per 100 ml leg, respectively. Transport of phenylalanine did not change significantly. We conclude that insulin promoted muscle anabolism, primarily by stimulating protein synthesis independently of any effect on transmembrane transport.

Intraoperative radiofrequency ablation or cryoablation for hepatic malignancies.

BACKGROUND The majority of patients with primary or metastatic malignancies confined to the liver are not candidates for resection because of tumor size, location, multifocality, or inadequate functional hepatic reserve. Cryoablation has become a common treatment in select groups of these patients with unresectable liver tumors. However, hepatic cryoablation is associated with significant morbidity. Radiofrequency ablation (RFA) is a technique that destroys liver tumors in situ by localized application of heat to produce coagulative necrosis. In this study, we compared the complication and early local recurrence rates in patients with unresectable malignant liver tumors treated with either cryoablation or RFA. PATIENTS AND METHODS Patients with hepatic malignancies were entered into two consecutive prospective, nonrandomized trials. The liver tumors were treated intraoperatively with cryoablation or RFA; intraoperative ultrasonography was used to guide placement of cryoprobes or RFA needles. All patients were followed up postoperatively to assess complications, treatment response, and local recurrence of malignant disease. RESULTS Cryoablation was performed on 88 tumors in 54 patients, and RFA was used to treat 138 tumors in 92 patients. Treatment-related complications, including 1 postoperative death, occurred in 22 of the 54 patients treated with cryoablation (40.7% complication rate). In contrast, there were no treatment-related deaths and only 3 complications after RFA (3.3% complication rate, P<0.001). With a median follow-up of 15 months in both patient groups, tumor has recurred in 3 of 138 lesions treated with RFA (2.2%), versus 12 of 88 tumors treated with cryoablation (13.6%, P<0.01). CONCLUSIONS RFA is a safe, well-tolerated treatment for patients with unresectable hepatic malignancies. This study indicates that (1) complications occur much less frequently following RFA of liver tumors compared with cryoablation of liver tumors, and (2) early local tumor recurrence is infrequent following RFA.

Down-regulation of Vascular Endothelial Growth Factor in a Human Colon Carcinoma Cell Line Transfected with an Antisense Expression Vector Specific for c-src

Vascular endothelial growth factor (VEGF) is implicated in the angiogenesis of human colon cancer. Recent evidence suggests that factors that regulate VEGF expression may partially depend on c-src-mediated signal transduction pathways. The tyrosine kinase activity of Src is activated in most colon tumors and cell lines. We established stable subclones of the human colon adenocarcinoma cell line HT29 in which Src expression and activity are decreased specifically as a result of a transfected antisense expression vector. This study determined whether VEGF expression is decreased in these cell lines and whether the smaller size and reduced growth rate of antisense vector-transfected cell lines in vivo might result, in part, from reduced vascularization of tumors. Northern blot analysis of these cell lines revealed that VEGF mRNA expression was decreased in proportion to the decrease in Src kinase activity. Under hypoxic conditions, cells with decreased Src activity had a <2-fold increase in VEGF expression, whereas parental cells had a >50-fold increase. VEGF protein in the supernatants of cells was also reduced in antisense transfectants compared with that from parental cells. In nude mice, subcutaneous tumors from antisense transfectants showed a significant reduction in vascularity. These results suggest that Src activity regulates the expression of VEGF in colon tumor cells.

Effectiveness of mastectomy by response to induction chemotherapy for control in inflammatory breast carcinoma

AbstractBackground: Controversy exists as to the treatment regimen necessary to best provide optimal local control for inflammatory breast carcinoma (IBC). This study was conducted to determine if mastectomy combined with radiotherapy offered any advantages over radiotherapy alone in patients with IBC who had been treated with doxorubicin-based combination chemotherapy. Methods: A retrospective review of 178 women treated for IBC on doxorubicin-based multimodality therapy protocols between January 1974 and September 1993 was performed. Clinical and histologic response to treatment, time to local recurrence, survival, and ultimate control of local disease were analyzed. Kaplan-Meier analysis was used to examine survival and relapse times, and Fishers exact test was used to test differences in treatment outcomes. Significance was determined at p≤0.05. Results: Median follow-up was 89 months (range 22 to 223 months). Locoregional disease persisted in seven patients and recurred in 44 patients who had been rendered disease free at a median time of 10 months. The mortality rate after a local recurrence (LR) was 98%, and all patients but one with LR developed systemic metastases. Response to induction chemotherapy influenced the incidence of LR, and the amount of residual disease found on histologic examination of mastectomy specimens was highly prognostic for local failure. Patients who underwent mastectomy in addition to radiotherapy had a lower incidence of LR than did patients who received radiotherapy alone (16.3% vs. 35.7%, p=0.015). Conclusions: The addition of mastectomy to combination chemotherapy plus radiotherapy improved local control in patients with IBC. The addition of mastectomy to chemotherapy plus radiotherapy improved distant disease-free and overall survival in patients with a clinical complete or partial response to induction chemotherapy. Patients who had no significant response to induction chemotherapy received no survival or local disease-control benefit from the addition of mastectomy to their treatment regimen. These patients should be considered for entry into clinical trials of new treatment regimens.

Laser optoacoustic imaging of breast cancer in vivo

A clinical prototype of the laser optoacoustic imaging system (LOIS) was employed for breast cancer detection and localization in patients with confirmed breast cancer and scheduled for radical mastectomy. The prototype LOIS used a single optical fiber for delivery of laser pulses, an arc shaped 32-element PVDF transducer array for ultrawide-band piezoelectric detection of optoacoustic signals and a single-channel data acquisition card for signal processing. The resonance ultrasound frequency of the 110 micrometers PVDF film was outside detectable range of ultrasound. Spatial resolution of the transducer array was slightly better than 1mm in radial direction and slightly worse than 1 mm in lateral direction. The system was optimized for contrast and sensitivity. Data acquisition, signal conditioning and image processing were significantly improved and optimized resulting in reduced image frame rate of 2 seconds employing 700 MHz Aphlon processor. The computer code for digital signal processing employed band-pass hyper-Gaussian filtering and denoising. An automatic recognition of the optoacoustic signal detected from the irradiated surface was implemented in order to visualize the breast surface and improve the accuracy of tumor localization. Radial back- projection algorithm was employed adopting combination of integration along spherical wavefronts and integration along planar wavefronts (as in Radon transform) for image reconstruction. The system performance was evaluated initially in breast tissue-like phantoms with embedded blood vessels. Clinical studies in breast cancer patients scheduled for surgical mastectomy were performed and compared with x-ray radiography, ultrasound and pathology reports.

Laser opto-acoustic imaging of the breast : Detection of cancer angiogenesis

First clinical prototype laser optoacoustic imaging system (LOIS) for breast cancer detection was designed and fabricated using a compact Nd:YAG laser, fiberoptic light delivery system, a linear array of 12 wide-band acoustic transducers, and a data acquisition card operated by computer with original signal processing and image reconstruction code. Initially images of small absorbing spheres were recorded in the milk with optical properties resembling those of the breast at the wavelength of 1064-nm. The system was optimized for contrast, sensitivity and axial (in-depth) resolution. The small number of acoustic transducers (12), which in turn was determined by the system cost and the time of image acquisition limited the lateral resolution of the images. Clinical ex-vivo studies on radical mastectomy specimens were performed and compared with x-ray radiography, MRI and ultrasound imaging. The results of our pilot clinical studies showed pronounced opto-acoustic contrast of ~300% between breast tumors and normal breast tissues. This contrast substantially exceeds any other endogenous tissue contrast currently utilized in clinical ultrasonography, MRI and x-ray mammography. Based on literature data and our gross observations of tumor cross-sections we hypothesize that the opto-acoustic contrast results primarily from increased optical absorption in the dense microvascularity of the tumors. In patients receiving radiotherapy, tumors were found to contain enhances concentration of dense highly scattering fiberotic tissue.

Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by activity of src kinase

BACKGROUND The c-src protooncogene encodes a protein tyrosine kinase, pp60c-src, that is a mediator in many signal transduction pathways. One pathway in which pp60c-src protein tyrosine kinase activity is implicated involves regulation of vascular endothelial growth factor (VEGF), an angiogenic factor important to neovascularization of growing tumors. Recently we demonstrated that decreased activity of pp60c-src in colon tumor cells contributes to decreased expression of VEGF. This study examined the relationship between pp60c-src activation, cell density, and VEGF production in a colon tumor cell line. METHODS Parental HT-29 colon adenocarcinoma cells and stable subclones created by transfection with c-src antisense and sense (control) expression vectors were plated under sparse (2 x 10(4) cells/cm2) and confluent (20 x 10(4) cells/cm2) conditions and grown for 36 hours. Protein and RNA were extracted from cells to determine pp60c-src levels, c-Src tyrosine kinase activity, and VEGF mRNA expression. RESULTS The pp60c-src kinase activity of HT-29 cells and control sense-transfected clones grown under confluent conditions was increased threefold to fivefold compared with cells grown under sparse conditions. In contrast, the ability of confluent culture conditions to increase pp60c-src activity was blunted in antisense transfectants. By regression analysis, VEGF expression was found to vary directly with pp60c-src levels (r2 = 0.886). CONCLUSIONS Cell density contributes to the regulation of c-src kinase activity and VEGF expression in HT-29 cells. When the steady-state level of pp60c-src is reduced in antisense transfectants, not only is the steady-state level of VEGF reduced, but the ability of confluence to stimulate pp60c-src activity and VEGF production is too. These data suggest that c-src may be an intermediary of both constitutive and inducible pathways for VEGF production in colon tumor cells.

Overexpression of focal adhesion kinase (p125FAK) in human colorectal carcinoma liver metastases: Independence from c-src or c-yes activation

AbstractBackground: p125FAK, pp60c-src, and pp62c-yes are protein tyrosine kinases that function in signaling pathways regulating cell adhesion, migration, and growth. The expression and tyrosine kinase activities of pp60c-src and pp62c-yes, and the expression of p125FAK are increased in colorectal tumor metastases relative to normal mucosa. This study investigates whether differences in the activation of pp60c-src and pp62c-yes in colorectal liver metastases correlated with differences in p125FAK expression and whether prognostic significance could be demonstrated from the extent of expression of p125FAK in metastases. Methods: Activities of pp60c-src and pp62c-yes were measured in the immune complex kinase assay. Relative levels of p125FAK, pp60c-src, and pp62c-yes were determined by immunoblotting. Results: p125FAK was overexpressed in 29 of 30 colorectal cancer liver metastases (range of two- to 195-fold increase compared with normal mucosa). The degree of overexpression of p125FAK was not a significant prognostic factor in survival. A differential activation of pp60c-src and pp62c-yes in colorectal carcinoma liver metastases was observed. However, overexpression of p125FAK was observed in metastases with either pp60c-src or pp62c-yes activated in colorectal carcinoma liver metastases. Conclusions: p125FAK overexpression appears to be a marker present in colorectal cancer cells with a metastatic phenotype. Furthermore, p125FAK overexpression is independent of pp60c-src or pp62c-yes activation in human colorectal carcinoma liver metastases.

The safety of helium for abdominal insufflation

AbstractBackground: A search for alternative methods of abdominal insufflation has been prompted by the fact that CO2 insufflation may cause acidosis, decreased cardiac output, increased systemic vascular resistance, and increased cardiac filling pressures. This study evaluates the safety and the cardiopulmonary effects of helium abdominal insufflation (HAI). Methods: Thirteen ASA class III and IV patients undergoing laparoscopic procedures were studied in a prospective, nonrandomized protocol using HAI. Cardiopulmonary parameters were measured before and after anesthetic induction and every 30 min during HAI. Abdominal insufflation pressure was initially 10 mmHg and was increased to 15 mmHg after 30 min. All measurements were repeated 15 min after deflation of the abdomen. Changes were evaluated by ANOVA. Results: No significant cardiopulmonary complications were observed. No patient developed hypercarbia or acidosis. Peak inspiratory pressure increased with HAI from 20 ± 1 to 34 ± 2 cm H2O (p < 0.0001). Cardiac index decreased (3.35 ± 0.19 vs 2.37 ± 0.19 l/min/m2; p= 0.0303) and systemic vascular resistance increased (1,123 ± 66 vs 1,406 ± 126 dyne · s/cm5; p= 0.0512) while cardiac filling pressures increased with insufflation to 15 mmHg. Conclusions: Minimal cardiac and pulmonary aberrations were observed. Helium was safe for abdominal insufflation and may be the insufflating agent of choice in patients with significant cardiopulmonary disease.

Optoacoustic tomography of breast cancer with arc-array transducer

The second generation of the laser optoacoustic imaging system for breast cancer detection, localization and characterization using a 32-element arc-shaped transducer array was developed and tested. Each acoustic transducer was made of 110-micrometers thick SOLEF PVDF film with dimensions of 1mm X 12.5mm. The frequency band of transducer array provided 0.4-mm axial in-depth resolution. Cylindrical shape of this 10-cm long transducer array provided an improved lateral resolution of 1.0 mm. Original and compact design of low noise preamplifiers and wide band amplifiers was employed. The system sensitivity was optimized by choosing limited bandwidth of ultrasonic detection 20-kHz to 2-MHz. Signal processing was significantly improved and optimized resulting in reduced data collection time of 13 sec. The computer code for digital signal processing employed auto- gain control, high-pass filtering and denoising. An automatic recognition of the opto-acoustic signal detected from the irradiated surface was implemented in order to visualize the breast surface and improve the accuracy of tumor locations. Radial back-projection algorithm was used for image reconstruction. Optimal filtering of image was employed to reduce low and high frequency noise. The advantages and limitations of various contrast-enhancing filters applied to the entire image matrix were studied and discussed. Time necessary for image reconstruction was reduced to 32 sec. The system performance was evaluated initially via acquisition of 2D opto-acoustic images of small absorbing spheres in breast-tissue-like phantoms. Clinical ex-vivo studies of mastectomy specimen were also performed and compared with x-ray radiography and ultrasound.