Pilot phase I/II personalized therapy trial for metastatic colorectal cancer: evaluating the feasibility of protein pathway activation mapping for stratifying patients to therapy with imatinib and panitumumab.

This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of c-Kit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT-mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms.

Novel measurements of mammary stem cells in human umbilical cord blood as prospective predictors of breast cancer susceptibility in later life.

BACKGROUND: The size of the breast stem-cell pool could underlie the intrauterine roots of breast cancer. We studied whether breast stem cells exist in umbilical cord blood and if they correlate with hematopoietic stem-cell measurements that have been positively associated with perinatal risk factors for breast cancer. SUBJECTS AND METHODS: We isolated mononuclear cells from umbilical cord blood of 170 singleton full-term pregnancies and determined, by reverse transcription polymerase chain reaction, the presence of genes of putative breast epithelial stem-cell/progenitor markers [including epithelial cell adhesion molecule (EpCAM), CD49f (α6-integrin), CD117 (c-kit receptor), CD24, and CD29 (ß1-integrin)]. By immunocytochemistry, we colocalized protein expressions of EpCAM+CD49f+, CD49f+CD24+, and CD24+CD29+. We correlated concentrations of putative breast stem-cell/progenitor subpopulations, quantified by flow cytometry, with concentrations of hematopoietic stem cells. RESULTS: Mammary stem-cell phenotypes were identified in umbilical cord blood. The measured EpCAM+ subpopulation was positively correlated with concentrations of CD34+ and CD34+CD38- hematopoietic stem cells (both P=0.006). Additionally, EpCAM+CD49f+ and CD49f+CD24+ subpopulations were positively correlated to the CD34+ cells (P=0.03 and 0.008, respectively). CONCLUSION: The positive association between measurable breast and hematopoietic stem cells in human umbilical cord blood suggests plausible mechanisms for a prenatal influence on breast cancer risk.

Correlation of umbilical cord blood haematopoietic stem and progenitor cell levels with birth weight: implications for a prenatal influence on cancer risk.

We examined the relation with birth weight and umbilical cord blood concentrations of haematopoietic stem and progenitor populations in 288 singleton infants. Across the whole range of birth weight, there was a positive relation between birth weight and CD34+CD38(-) cells, with each 500 g increase in birth weight being associated with a 15.5% higher (95% confidence interval: 1.6-31.3%) cell concentration. CD34+ and CD34+c-kit+ cells had J-shaped relations and CFU-GM cells had a U-shaped relation with birth weight. Among newborns with >or=3000 g birth weights, concentrations of these cells increased with birth weight, while those below 3000 g had higher stem cell concentrations than the reference category of 3000-3499 g. Adjustment for cord blood plasma insulin-like growth factor-1 levels weakened the stem and progenitor cell-birth weight associations. The positive associations between birth weight and stem cell measurements for term newborns with a normal-to-high birth weight support the stem cell burden hypothesis of cancer risk.

Validity of BMI as a measure of obesity in Australian white Caucasian and Australian Sri Lankan children.

BACKGROUND: Body mass index (BMI) is used to diagnose obesity. However, its ability to predict the percentage fat mass (%FM) reliably is doubtful. Therefore validity of BMI as a diagnostic tool of obesity is questioned. AIM: This study is focused on determining the ability of BMI-based cut-off values in diagnosing obesity among Australian children of white Caucasian and Sri Lankan origin. SUBJECTS AND METHODS: Height and weight was measured and BMI (W/H2) calculated. Total body water was determined by deuterium dilution technique and fat free mass and hence fat mass derived using age- and gender-specific constants. A %FM of 30% for girls and 20% for boys was considered as the criterion cut-off level for obesity. BMI-based obesity cut-offs described by the International Obesity Task Force (IOTF), CDC/NCHS centile charts and BMI-Z were validated against the criterion method. RESULTS: There were 96 white Caucasian and 42 Sri Lankan children. Of the white Caucasians, 19 (36%) girls and 29 (66%) boys, and of the Sri Lankans 7 (46%) girls and 16 (63%) boys, were obese based on %FM. The FM and BMI were closely associated in both Caucasians (r=0.81, P<0.001) and Sri Lankans (r=0.92, P<0.001). Percentage FM and BMI also had a lower but significant association. Obesity cut-off values recommended by IOTF failed to detect a single case of obesity in either group. However, NCHS and BMI-Z cut-offs detected cases of obesity with low sensitivity. CONCLUSIONS: BMI is a poor indicator of percentage fat and the commonly used cut-off values were not sensitive enough to detect cases of childhood obesity in this study. In order to improve the diagnosis of obesity, either BMI cut-off values should be revised to increase the sensitivity or the possibility of using other indirect methods of estimating the %FM should be explored.

Impact of ethnicity upon body composition assessment in Sri Lankan Australian children.

OBJECTIVES: Obesity is a disease with excess body fat where health is adversely affected. Therefore it is prudent to make the diagnosis of obesity based on the measure of percentage body fat. Body composition of a group of Australian children of Sri Lankan origin were studied to evaluate the applicability of some bedside techniques in the measurement of percentage body fat. METHODS: Height (H) and weight (W) was measured and BMI (W/H(2)) calculated. Bioelectrical impedance analysis (BIA) was measured using tetra polar technique with an 800 microA current of 50 Hz frequency. Total body water was used as a reference method and was determined by deuterium dilution and fat free mass and hence fat mass (FM) derived using age and gender specific constants. Percentage FM was estimated using four predictive equations, which used BIA and anthropometric measurements. RESULTS: Twenty-seven boys and 15 girls were studied with mean ages being 9.1 years and 9.6 years, respectively. Girls had a significantly higher FM compared to boys. The mean percentage FM of boys (22.9 +/- 8.7%) was higher than the limit for obesity and for girls (29.0 +/- 6.0%) it was just below the cut-off. BMI was comparatively low. All but BIA equation in boys under estimated the percentage FM. The impedance index and weight showed a strong association with total body water (r(2)= 0.96, P < 0.001). Except for BIA in boys all other techniques under diagnosed obesity. CONCLUSIONS: Sri Lankan Australian children appear to have a high percentage of fat with a low BMI and some of the available indirect techniques are not helpful in the assessment of body composition. Therefore ethnic and/or population specific predictive equations have to be developed for the assessment of body composition, especially in a multicultural society using indirect methods such as BIA or anthropometry.

Reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells.

Microscopic infarcts develop within the livers of athymic nude mice during the first 24 h after human colorectal carcinoma (CRC) cells arrest within hepatic sinusoids. Because these regions are reperfused, essentially all weakly metastatic clone A and MIP-101 CRC cells die, whereas many highly metastatic CX-1 CRC cells survive. Because hepatic sinusoidal endothelial cells kill tumor cells in vitro by producing nitric oxide, superoxide anion, and other reactive oxygen and nitrogen species, our purpose was to determine whether reoxygenation of ischemic hepatic cultures in vitro forms toxic oxygen and nitrogen radicals that kill weakly but not highly metastatic CRC cells. CRC cells (10(7)) were labeled with rhodamine-dextran and calcein AM, cultured with cells from one mouse liver in a rotating suspension culture system for up to 24 h, and the metabolic activity of the CRC cells was determined. Liver fragments oxygenated normally before harvest were not toxic to either CRC cell line, but coculture with liver made ischemic by a 3-min ligation of the portal vein and hepatic artery in vivo before harvest and then cultured in oxygenated medium killed 50-70% of weakly metastatic clone A and MIP-101 cells at 24 h but <15% of highly metastatic CX-1 cells. Inhibition of nitric oxide synthase, addition of exogenous superoxide dismutase, but not catalase or hypoxia, during coculture blocked the killing of weakly metastatic CRC cells. Thus, reoxygenation of hepatic parenchymal and nonparenchymal cells after ischemia may form toxic species that eliminate weakly metastatic CRCs within 24 h of their arrest in the liver.

Carcinoembryonic antigen facilitates experimental metastasis through a mechanism that does not involve adhesion to liver cells.

Carcinoembryonic antigen (CEA) injected intravenously into athymic nude mice increases the ability of weakly metastatic human colorectal carcinoma (CRC) cells to colonize liver in an experimental metastasis assay. Since CEA acts as an intercellular adhesion molecule in vitro, several investigators have postulated that this facilitation of experimental metastasis may be mediated through adhesion between CEA on CRC and CEA-binding proteins on Kupffer or other cells lining the hepatic sinusoid. The present work tested this postulate both by intravital fluorescence videomicroscopy in vivo and in adhesion assays in vitro to enriched populations of Kupffer cells and hepatic sinusoidal endothelial cells (SEC). The data indicate that CEA expression does not effect adhesion to enriched Kupffer cells or SEC in vitro. These data suggest that CEA enhances liver colonization through another mechanism, possibly one that involves modulation of the hepatic response to tumor cell implantation.

Role of nitric oxide and superoxide anion in elimination of low metastatic human colorectal carcinomas by unstimulated hepatic sinusoidal endothelial cells.

Human colorectal carcinoma (CRC) cell survival for the first 24 h after implantation in the hepatic sinusoid determines its potential to colonize the liver. Nearly 10-fold more highly metastatic CX-1 cells survive within the livers of nude mice 24 h after intrasplenic injection than weakly metastatic clone A cells. Because CRCs contact sinusoidal endothelial cells (SECs) during implantation, we sought to determine whether SECs were more toxic to clone A than to CX-1 cells. When 2 x 10(4) vital dye-labeled CRC cells were added to murine SEC monolayers, more than 30% of clone A cells lost calcein AM fluorescence compared to fewer than 5% of CX-1 cells after 24 h of coculture with SECs. Kupffer cells did not mediate this effect, because neither enriched Kupffer cells nor SECs treated with a Kupffer cell inhibitor altered the SEC-mediated toxic effect to clone A cells. Pretreatment with a nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, superoxide dismutase, or dexamethasone, blocked SEC-mediated toxicity to clone A cells, whereas calcium chelation and catalase did not. In addition, clone A cells were more sensitive to a superoxide donor, 3-morpholinosydnonimine N-ethylcarbamide, than were CX-1 cells, and neither cell line was sensitive to sodium nitroprusside, a nitric oxide donor. Thus, unstimulated murine SECs produce reactive oxygen species that are selectively toxic to weakly metastatic clone A cells. This may be a mechanism by which host liver cells eliminate weakly metastatic neoplastic cells.

In vivo induction of murine cytokine production by carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) may promote experimental metastasis through production of cytokines. The effect of systemic CEA on the production of proinflammatory cytokines was investigated in mice and compared to levels induced by lipopolysaccharide (LPS). Serum concentrations of interleukin (IL)-6 peaked 1 h after an i.v. CEA injection of 40 microg/mouse to 37-54% of the maximal level induced by a 1 microg/mouse injection of LPS in both normal and immunoincompetent mice. The CEA induction of IL-6 was a specific response, because the peptide PELPK (the pentapeptide on CEA that is the ligand for the CEA receptor on Kupffer cells) conjugated to albumin induced 30% of the maximal CEA response for IL-6, whereas the specificity control PELGK-conjugated albumin did not. IL-1alpha and tumor necrosis factor (TNF)-alpha levels after i.v. injection of CEA were only 3-5% of those induced by LPS. The IL-6 responses of mice pretreated with 100 microg/kg genistein were decreased by more than 40%. However, genistein inhibited the TNF-alpha response to LPS by 46% but increased the CEA-induced response by 300%. When murine Kupffer cells were stimulated with LPS or CEA in vitro, LPS increased tyrosine phosphorylation of a Mr 30,000 protein, whereas CEA decreased phosphorylation of a Mr 60,000 protein and did not increase phosphorylation of the Mr 30,000 protein. Thus, i.v. CEA stimulates production of IL-6 and TNF-alpha after binding to Kupffer cells through signal transduction pathways that appear to be different from those stimulated by LPS.

Breast cancer screening: early recognition.

The epidemic of breast cancer and the availability of tools to increase early detection and decrease mortality require care and diligence on the part of every physician directly caring for patients. With the widespread implementation of managed care, primary care physicians are in the best position to recommend breast cancer screening. Educational programs to emphasize the primary care physician's role in breast cancer prevention and detection, to improve physical examination skills, and to implement office systems to facilitate screening should be available to primary care physicians, and may have a major impact on breast cancer mortality in the future.

Attitudes toward risk factor behavior of relatives of cancer patients.

BACKGROUND: Targeted health promotion requires an identifiable subpopulation which is accessible, at increased risk, receptive to input, and receptive to change. Relatives of recently diagnosed cancer patients may meet these criteria and have not previously been investigated as recipients of preventive education regarding smoking and diet. METHODS: This study investigates these factors, beliefs regarding perceived susceptibility to cancer, and attitudes toward behavior change in 101 relatives of 50 patients with smoking-related cancers, breast cancer, and other diet-related cancers. Congruence of attitudes between patients and relatives, another possible factor in changing health behaviors, also was assessed. RESULTS: Access to relatives of patients was very high, as was their willingness to discuss these issues (99% of relatives contacted participated in the survey). Relatives' ratings of relevant risk factors were generally higher than those of patients; ratings of their own vulnerability were moderate. Within diagnostic groups, there was high concordance of belief between patients and relatives for certain types of risk, such as heredity for breast cancer (r = 0.81) and smoking for smoking-related cancers (r = 0.52), but not for dietary factors. CONCLUSIONS: The high level of access suggests that relatives may be receptive to discussing issues of behavior risk and change. They are at least as aware as patients of cancer risk factors. Spontaneous behavior change was very low. They may therefore be good candidates for targeted health promotion regarding cancer risk.

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia.

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.

Effect of a mild exercise program on myocardial function and the development of hypertrophy.

Cardiac function and the development of myocardial hypertrophy were studied in rats conditioned by an exercise program consisting of 8 wk of running on a treadmill. At the end of the training period a group of exercised and sedentary rats was subjected to hemodynamic evaluation under general anesthesia. Except for a slight elevation in the heart rates of the exercised animals there were no significant differences between the exercised and sedentary rats at rest. Following an increase in afterload or a period of hypoxia, the cardiac index of the exercised animals remained significantly higher than that of the sedentary controls. These differences were related to changes in stroke volume. Another group of exercised and sedentary animals underwent either constriction of the ascending aorta or a sham operation. Sedentary rats developed significant hypertrophy at 3 days but had no hypertrophy at 1 day after aortic constriction. Exercised rats, however, developed significant myocardial hypertrophy by 1 day after pressure overload. These data suggest that the heart from an exercised animal is better able to tolerate increases in afterload and hypoxia and can respond with compensatory myocardial hypertrophy more rapidly than the heart of a sedentary animal.