Prognostic significance of biologic factors in squamous cell carcinoma of the esophagus.

BACKGROUND: Esophageal carcinoma is one of the most lethal tumors. Therefore, it is important to identify prognostic factors for patients with this disease. The objective of this study was to clarify the relation between clinicopathologic and biologic factors in esophageal carcinoma and to determine the prognostic significance of different biologic factors. METHODS: DNA ploidy pattern, Ki-67 labeling index (LI), and cyclin D1 and p53 protein expression were examined and detailed pathologic examinations were conducted on tumors from 53 patients (46 males and 7 females with a mean age of 66 years [range, 47-85 years]) with surgically resected esophageal squamous cell carcinoma and the prognostic value of these factors was evaluated. RESULTS: Of the 53 esophagus carcinomas examined, 26 (49%) were classified as DNA diploid. The mean Ki-67 LI was 45 +/- 4. 9% (range, 10.5-86.1%). p53 expression was detected in 38 of the carcinomas (71.7%) and cyclin D1 expression was detected in 35 (66%). Various prognostic factors were examined using the Cox stepwise regression model, four of which were found to correlate with overall survival: tumor size (P = 0.0346), lymph node status (P = 0.0384), Ki-67 LI (P = 0.0161), and p53 expression (P = 0.001). Lower Ki-67 LI and a lower rate of p53 expression were detected in the long term survival group (> 3 years) compared with the short term survival group (P = 0.00045 and P = 0.0023, respectively). CONCLUSIONS: The biologic factors of Ki-67 LI and p53 expression, as well as clinicopathologic factors, may be used as independent prognostic factors for patients with esophageal carcinoma. However, the results of the current study do not support cyclin D1 expression as a prognostic factor.

Distribution and excretion of radiolabelled tert-butylhydroquinone in Fischer 344 rats.

Uniformly 14C-ring-labelled tert-butylhydroquinone (TBHQ) was diluted with non-radioactive TBHQ and administered orally (for excretion studies) to Fischer 344 rats. An average of 72.9% and 10.6% of the administered radioactivity was recovered in the urine and faeces, respectively, of male rats, and 77.3% and 8.2% in the urine and faeces, respectively, of female rats in 4 days. No significant sex-related differences were found in either excretion, tissue distribution or urinary metabolites of TBHQ-derived radiolabel. For distribution studies, intraperitoneal doses were administered to female rats, and tissue levels of radiolabel were determined at various times after dosing. The parent compound quickly disappeared from tissue in vivo. The highest concentrations of radiolabel were found in the liver and kidneys. The urinary metabolites consisted of conjugated TBHQ and unidentified polar substance(s).

A case of transverse colon cancer secondarily involving the liver, duodenum, and pancreas.

A 68-year-old woman presented with transverse colon cancer invading the liver, duodenum, and pancreas. The patient underwent a curative resection including a right hemicolectomy, partial hepatectomy, and pancreaticoduodenectomy (PD). The pathological examination showed adenocarcinoma of the colon with a direct extension into the duodenum, liver, and pancreas. Several lymph nodes were also involved. The patient is still alive and disease-free 2 years and 6 months after the operation. This case illustrates that even in patients with locally advanced colon cancer, a favorable prognosis can be obtained by aggressive surgery incorporating the resection of the adjacent involved organs.

Distribution, metabolism and excretion of pentachloroanisole in the beagle dog and miniature pig.

Tissue distribution, excretion and metabolism studies of pentachloroanisole (PCA), an environmental metabolite of pentachlorophenol (PCP), were conducted in the beagle dog and miniature pig following single oral doses (25 mg/kg) of radiolabelled PCA. PCA was readily demethylated by both species, with a half-life of 5-8 min. The resultant PCP was the major metabolite in dogs and pigs. In the dog, an average of 21.9% of the administered radiolabel was excreted in the urine and 62.3% in the faeces during a 7-day period. Of the tissues analysed, an average of 3.2% of the radiolabel remained in the liver, and blood and muscle accounted for averages of 3.0 and 2.3%, respectively, of the dose. Free and conjugated PCP were found in the urine of dogs; no PCA or tetrachlorohydroquinone (TCH) were found. In dog faeces, PCP and a trace of polar material were observed; no PCA was excreted in dog faeces. In the miniature pig, an average of 25.8% of the administered radiolabel was excreted in the urine and 32.0% in the faeces during a 2-wk period. An average of 4.4% of the radiolabel was found in the liver, 8.8% in the blood, 7.1% in the muscle and 6.4% in the fat. In pig urine, PCP and conjugated PCP were the only metabolites observed; no PCA or TCH was found. Pig faeces contained a trace of unchanged PCA; PCP and polar metabolites were also found. Since pig tissues retained a sizeable residue 2 wk after a single dose of PCA, various agents were used in an attempt to decrease the tissue level of radiolabel in pigs; anion exchange resin was found to be the most effective.

Disposition and metabolism of radiolabelled pentachloroanisole in rats and rabbits.

Male Sprague-Dawley rats and New Zealand White rabbits were administered 14C-labelled pentachloroanisole (PCA) in corn oil by gavage as single doses of 25 mg/kg and were then placed in individual metabolism cages for as long as 4 days. Peak blood level of radioactivity occurred 6 hr after administration of the dose to rats and between 3 and 4 hr in rabbits; the blood elimination half-life ranged from 8 to 15 hr in rats and averaged 6 hr in rabbits. Rats excreted an average of 54.2% of the administered radiolabel in the urine and 32.4% in the faeces during the 96 hr following the dose; rabbits excreted an average of 84.2 and 13.1% of the radiolabel in the urine and faeces, respectively, during this time. Examination of the metabolites in the rat showed that 60% of the urinary radioactivity was attributable to tetrachlorohydroquinone (TCH), 3% to free pentachlorophenol (PCP) and 29% to conjugated PCP; faecal metabolites were PCP (85.7%), TCH (4.3%) and polar metabolite(s) (10%). In the rabbit, 58% of the urinary radioactivity was attributable to TCH, 8% to free PCP and 34% to conjugated PCP. Faecal metabolites consisted of PCP and conjugated material.

Tissue distribution and excretion of 14C-labelled cinnamic aldehyde following single and multiple oral administration in male Fischer 344 rats.

14C-labelled cinnamic aldehyde (CNMA) was given as a single oral dose, or 24 hr after multiple oral administration of non-radioactive CNMA for 7 days at 24-hr intervals, to male Fischer 344 rats at dose levels of 5, 50 or 500 mg/kg body weight. Residues of radioactive CNMA were measured. After the single dose radioactivity was distributed primarily in the gastro-intestinal tract, the kidneys and the liver of the rats. The radiolabel was excreted mainly in the urine, and at 24 hr 85.1, 84.2 and 81.2% of the administered radiolabel was recovered in the urine at the 5, 50 and 500 mg/kg dose levels, respectively. Faecal excretion of radiolabel at 24 hr for the 5, 50 and 500 mg/kg doses was 5.1, 4.0 and 3.2% of the administered dose, respectively. At all dose levels, a small amount of the dose was distributed to the fat and was easily measured in animals killed 3 days after dosing at the 50 or 500 mg/kg dose levels. Following multiple oral administration, similar tissue distribution and excretion patterns of radiolabel were found at the three dose levels. After 24 hr the administered radiolabel was distributed mainly to the fat, liver and gastro-intestinal tract. At 24 hr, recoveries of the radiolabel in the urine were 80.4, 80.6 and 81.9% of the dose for the 5, 50 and 500 mg/kg dose levels, respectively. Faecal excretion of radiolabel after multiple dosing at 24 hr accounted for 6.3, 6.9 and 4.5% of the administered radioactivity at the 5, 50 and 500 mg/kg dose levels, respectively. The major metabolic pathway of CNMA for all single and the 5 and 50 mg/kg multiple dose levels in this species of rat was found to be degradation to benzoic acid through beta-oxidation and excretion in the urine mainly as hippuric acid, with much smaller amounts of benzoic and cinnamic acids. At the multiple dose level of 500 mg/kg, benzoic acid was the major urinary metabolite, indicating that in the Fischer 344 male rat at this relatively high oral dose level the detoxification of CNMA proceeds differently and an alternative metabolic pathway is proposed.

Time required to achieve homogeneity of test substances added to dog feed.

The homogeneity of test substances in a carrier (animal feed) is a critical factor in conducting long-term feeding studies in laboratory animals. A method for determining the adequate amount of mixing to achieve homogeneity by a mixer of the type described has been determined when 2 distinctly different compounds are added to ground dog feed. Nicotinic acid and butylated hydroxyanisole at a concentration of 1% were separately mixed with the dog feed for 15, 30, 45, 60, and 120 min to determine optimum mixing time. Test portions were taken from 4 different sampling sites at each time period and analyzed in duplicate for the added substance. Four batches were prepared and the results were aggregated. Very little interbatch variability was observed. The variance of the average values from the 4 sampling sites at each time period was calculated and used as a simple, crude, but effective numerical quantity to monitor the approach to homogeneity of the mixture.

Excretion and tissue distribution of 14C-labelled 8-methoxypsoralen in beagle dogs and miniature pigs.

Male beagle dogs and miniature swine were given 14C-labelled 8-methoxypsoralen (8-MOP) as a single oral dose (10 mg/kg body weight). In dogs, there appeared to be wide variability in 8-MOP absorption as indicated by the broad range of percentages of radioactivity recovered from urine and faeces over a 4-day period (3.6-24.7% in urine; 47.3-94.9% in faeces); mean recovery values were 13.6% in the urine and 68.6% in the faeces. In pigs, considerable variability in absorption was also evident, but not to the extent of that seen in dogs. Based on the fraction of the dose recovered in the urine, the absorption of 8-MOP was greater in pigs; the proportion of the dose recovered in urine over a 7-day period ranged from 25.8 to 57.8%. Faecal recovery ranged from 15.4 to 49.0% of the dose. Mean recovery values in pigs were 45.4% in urine and 35.6% in faeces. Most of the 8-MOP was cleared from the bodies of dogs and pigs in a few days, and little 8-MOP residue was sequestered in any of the tissues examined in either species. Small amounts of an 8-MOP-related substance remained in the liver and blood for as long as 4 days in dogs and 7 days in pigs.

Comparative tissue distribution and excretion of [1-14C]acrylamide in beagle dogs and miniature pigs.

Male beagle dogs and miniature pigs were given acrylamide in the diet for 3-4 wk at a dosage of 1 mg/kg/day. They were then given [1-14C]acrylamide as a single oral dose of 1 mg/kg. The animals were killed 6 hr or 1, 2, 4 or 14 days after administration of the radioactive compound and tissues were analysed for radioactivity. The radiolabelled material was distributed to a major extent in muscle tissue in both species (31-35% of the dose at 6 hr and 5-7% at 14 days). Although the nervous system is the primary target for acrylamide monomer toxicity, less than 1% of the administered 14C was found in the brain in both species. No neurotoxic signs were evident during the exposure period at the dosage used. Analysis of discrete areas of the brain for radioactivity revealed that the levels of penetration of [1-14C]acrylamide in brain paralleled the vascularization pattern of the tissues. Approximately 60% of the administered radiolabel was excreted in the urine in both species and smaller amounts were excreted in the faeces. However, recovery in the faeces was higher in pigs (c. 25%) than in dogs (c. 7%) and this and the considerably higher levels demonstrated in the gastro-intestinal tract of the pigs indicated that the absorption of acrylamide was more rapid and more extensive in dogs than in pigs.

Effect of subchronic dietary administration of butylated hydroxyanisole on canine stomach and hepatic tissue.

Beagle dogs, 29 males and 30 females, were assigned to feeding groups of 0, 1.0 and 1.3% butylated hydroxyanisole (BHA) for 180 days. Animals were observed daily for physical signs of pharmacological or toxicological effect. Except for the production of a reddish-brown urine, no physical signs attributable to BHA administration were observed. Both food consumption and body-weight gain were reduced in the BHA-treated animals. Fifty-one animals completed the study. At termination, tissues were examined for gross BHA-related effects, and specimens were taken for enzyme analysis, light microscopy, electron microscopy and morphometric analysis. The liver showed a significant weight increase over the control in both sexes at both BHA dose levels. Ultrastructural examination of the liver of BHA-treated animals revealed proliferation of smooth endoplasmic reticulum and hepatocytic cytoplasmic myelinoid bodies. Enzyme analysis of hepatic tissue showed a significant increase in mixed-function oxidases, UDP glucuronyl transferase, glutathione S-transferase and epoxide hydratase in the BHA-treated dogs compared with the controls. Light microscopy revealed no proliferative/hyperplastic lesions of the stomach/gastric epithelium. Electron microscopic examination of the lower oesophagus and stomach specimens from representative animals from each treatment group failed to reveal any treatment-related effect as compared with controls.

Maternal-foetal distribution studies in late pregnancy. II. Distribution of [1-14C]acrylamide in tissues of beagle dogs and miniature pigs.

[carbonyl-14C]Acrylamide was administered iv as a single dose (5 mg/kg) to pregnant beagle dogs and miniature pigs late in gestation. After a 2-hr equilibration period, the animals were killed and foetuses were removed for determination of the amount of radioactivity in maternal and foetal tissues. In total, six dog litters (33 foetuses) and seven pig litters (45 foetuses) were examined. In dogs, acrylamide was distributed readily to both maternal and foetal tissues with a placental distribution factor of 17.7%. The blood/brain distribution factor was insignificant (5.9%) in maternal dogs and 0% in the foetuses. Maternal liver was the largest depot of the administered acrylamide in the dog, followed by the maternal kidney. In pigs, the placental distribution factor was 31%, and the blood/brain distribution factor was insignificant in both maternal and foetal pigs. Liver and kidney of maternal pigs also contained the greatest amount of radioactivity. Although there appears to be some placental protection of the foetuses from the xenobiotic in the maternal circulation, foetal brain would be exposed to the effect of any acrylamide present in the foetal circulation, since the foetuses of both species had blood/brain distribution factors that were either small or zero, reflecting the absence of a blood-brain barrier.

Maternal-foetal distribution studies in late pregnancy. I. Distribution of [N-methyl-14C]betaine in tissues of beagle dogs and miniature pigs.

[N-Me-14C]Betaine was administered iv as a single dose (5 mg/kg) to pregnant beagle dogs and miniature pigs late in gestation. Two hr after administration of the radiolabel, when the compound was in equilibrium, the dams were killed and the foetuses were removed for determination of the radioactivity in maternal and foetal tissues. Eight litters of dogs (56 foetuses) and four litters of pigs (30 foetuses) were examined. The distribution of betaine in both species showed distinct differences between maternal and foetal tissues, indicating definite placental barriers; the placental distribution factor was estimated to be 52.3% in dogs and 97.8% in pigs. The blood/brain distribution factor was 84.6% in maternal dogs, 89% in maternal pigs, 65.7% in foetal dogs and 0% in foetal pigs. In the dog, maternal liver was the largest depot of the administered betaine, followed by foetal liver. Foetal heart, lung and kidney tissues also incorporated radiolabelled betaine. The highest concentrations of betaine in the pig were found in maternal kidney and liver.

Distribution of 14C-labelled acrylamide and betaine in foetuses of rats, rabbits, beagle dogs and miniature pigs.

[14C]Acrylamide and [14C]betaine hydrochloride were administered in a single iv dose to pregnant rats, rabbits, beagle dogs and miniature pigs late in gestation (1-2 days before expected parturition). Dosages used were 10 mg/kg for rats and 5 mg/kg for the other species. The compounds were allowed to equilibrate in the animal (for 1 hr in rats and for 2 hr in the other species); the dam was then killed and the foetuses were removed by caesarean section. Each foetus was weighed and analysed for radioactivity, either by homogenization of the whole foetus (rat and rabbit) or by determining separately the radioactivity in individual organs and tissues (dog and pig). Foetal uptake of the polar compound betaine hydrochloride was much lower than that of the more lipophilic acrylamide. The sex of the foetus did not appear to affect uptake of either compound. There were no significant differences in total uptake of isotope attributable to the position of the foetus within the uterus in any of the four species given either acrylamide or betaine. Similarly, uterine position did not affect the uptake of acrylamide or betaine by individual tissues of foetal dogs or pigs. Since the distributions of 14C-labelled acrylamide and betaine hydrochloride were essentially uniform throughout a litter, it would not be necessary to sample all of the members of a litter to obtain a representative picture of foetal distribution.

Blood levels of caffeine and results of fetal examination after oral administration of caffeine to pregnant rats.

Pregnant FDA-strain Osborne-Mendel rats were administered repeated doses of caffeine by oral intubation (gavage) and by administration in the drinking water (ad libitum sipping). When [1-methyl-14C]caffeine was administered at a dosage of 80 mg per kg per day by ad libitum sipping on days 12 to 15 of gestation, the amounts of radioactivity in blood were variable; the highest level on day 12 was 0.2% of the dose per ml of blood. The highest blood level of caffeine observed during a 24-h sampling period averaged 5.7 micrograms ml-1. When [14C]caffeine was administered by gavage at a dosage of 80 mg kg-1 on day 12, the blood level of radioactivity reached a peak of 0.4% of the dose per ml of blood and declined rapidly thereafter. The highest amount of caffeine observed in blood averaged 63.1 micrograms ml-1, 1 h after gavage. The overall blood elimination half-life of radioactivity in pregnant rats treated by gavage was 2.6 h, and the half-life of caffeine in blood was 1.7 h. The levels of radioactivity in the fetus and maternal muscle per unit weight were comparable after each method of administration. A comparison of autopsy results from both groups indicated that resorptions were increased when compared with rats that did not receive caffeine; this effect was more marked in the gavage group than in the ad libitum sipping group. Ectrodactyly was observed only in offspring of the gavage group. The incidences of ectrodactyly or resorptions did not appear to be directly related to nutrition or fluid intake.

Time required to achieve homogeneity in swine feed mixtures.

FD&C Red No. 3 was mixed with 20 kg pig feed to give a concentration of 0.1%. A mixing time of 30 min was sufficient to achieve homogeneity for this mixture. For larger amounts or more flocculent types of additives, a longer time may be required. Ammoniated glycyrrhizin was mixed with 8 separate batches of pig feed at a concentration of 1%; 1 h was sufficient mixing time.

Early pregnancy testing: a comparison of hemagglutination inhibition and radioreceptor assay.

Two methods of early pregnancy testing, Neocept hemagglutination inhibition assay, and Biocept-G radioreceptor assay, were compared using 508 female patients from whom both urine and serum specimens were submitted for routine testing. A conventional urine HAI test was also used to support results. Neocept and biocept-G were found to be equally effective, detecting 98% and 96% respectively of pregnant patients. Laboratory personnel determined the HAI methods to be simpler and more convenient, consuming less time and requiring less overhead.

PIP: Early pregnancy was tested in 508 women with 3 different types of tests: Neocept, a new hemagglutination inhibition assay (HAI), Pregnosticon Accuspheres, a conventional HAI test, and Biocept-G, a radioreceptor assay (RRA). The 1st 2 tests indicate the presence of human chorionic gonadotropin in the urine; the 3rd test indicates the presence of human chorionic gonadotropin in blood serum. In this series, there were 241 confirmed pregnancies; Neocept detected 98%, Biocept-G 96%, and Pregnosticon 90%. It seems apparent that Neocept and Biocept-G are of the same order of clinical reliability; pregnosticon is somewhat less effective, and therefore will be most useful with specimens collected after the missed period when human chorionic gonadotropin levels have risen into the sensitivity limits of this test. Given the equal effectiveness for the RRA and the new HAI test, it must be kept in mind that the Neocept procedure requires only 5 steps to RRAs 9, and that it requires less time for set up. The RRA test requires segregate storage and test areas, special disposal, and can be possibly hazardous.