The Cooperative Breast Cancer Tissue Resource: archival tissue for the investigation of tumor markers.

Investigators continue to search for reliable markers of prognosis of breast cancer. For many analyses, laboratory techniques permit the use of archival paraffin-embedded tissue collected years previously and readily linked to clinical and follow-up information. Laboratory investigators have often expressed the need for such a tissue resource. We have developed a publicly available resource of archival breast cancer specimens. The pathological material has been collected and reviewed by investigators at four institutions and currently includes breast cancer specimens from more than 9300 cases. Institutional pathologists reviewed slides and blocks using a common protocol and coding scheme. Clinical information and details of follow-up came from data routinely collected by the institutions' cancer registries. Coded data are maintained centrally in a single database. A subset of the data may be searched on the World Wide Web to determine the availability of cases with specified characteristics. The material collected by this Cooperative Breast Cancer Tissue Resource is generally representative of breast cancer diagnosed in community hospital settings in the United States. Seventy-two percent of the living cases have been followed for at least 5 years, and follow-up status is updated regularly. Interested laboratory investigators may apply to the Resource for the use of these tissues. This Resource is proving valuable to laboratory investigators who require large numbers of specimens for validation studies of prognostic markers of breast cancer.

Reproducibility of p53 immunohistochemistry in bladder tumors. National Cancer Institute, Bladder Tumor Marker Network.

The National Cancer Institute Bladder Tumor Marker Network conducted a study to evaluate the reproducibility of immunohistochemistry for measuring p53 expression in bladder tumors. Fifty paraffin blocks (10 from each of the five network institutions) were chosen at random from among high-grade invasive primary bladder tumors. Two sections from each block were sent to each laboratory for staining and scoring, and then all sections were randomly redistributed among the laboratories for a second scoring. Intra- and interlaboratory reproducibility was assessed with regard to both staining and scoring. For overall assessments of p53 positivity, the results demonstrated that intralaboratory reproducibility was quite good. Concordance across the five participating laboratories was high for specimens exhibiting no or minimal nuclear immunostaining of tumor cells or high percentages of tumor cells with nuclear immunoreactivities. However, there was a reduced level of concordance on specimens with percentages of stained tumor cells in an intermediate range. The discordancies were due mainly to staining differences in one of the five laboratories and scoring differences in another laboratory. These results indicate that some caution must be used in comparing results across studies from different groups. Standardization of staining protocols and selection of a uniform threshold for binary interpretation of results may improve assay reproducibility between laboratories.

Influence of triiodothyronine withdrawal time on 131I uptake postthyroidectomy for thyroid cancer.

Radioiodine uptake by thyroid remnants and metastases postthyroidectomy for thyroid cancer is increased by withdrawing thyroid hormone, which raises TSH levels. The minimal withdrawal time for maximal uptake is unknown. Therefore, we performed 33 studies in 27 patients after 2 weeks and again after 4 weeks of T3 withdrawal. We examined cervical (or pulmonary) uptake and whole body scanning at 48 h and whole body retention at 48, 72, and 96 h after radioiodine. In 4 studies, only physiological nonthyroidal activity was seen on both scans. Cervical uptake was low in these 4 studies. Of the remaining 29 studies with thyroid activity on both scans, 4 had high cervical uptakes after 2 weeks, which decreased by 4 weeks to less than 50% of the 2 week value. The same trend was seen in whole body retentions. In 2 studies, the uptake increased at 4 weeks compared to that at 2 weeks, but the change was small and was reflected in whole body retention of only 1 of these subjects. In 23 studies, including 6 with metastatic disease, the individual uptakes and whole body retentions were similar after 2 and 4 weeks. The mean uptakes and retentions also did not differ despite significantly higher (P less than 0.001) TSH values at 4 weeks. All definite areas of localization of radioactivity seen on the scans after 4 weeks were seen after 2 weeks. Therefore, radioiodine uptake, scanning, and therapy should be performed after 2 weeks of T3 withdrawal when patients are minimally hypothyroid. Serum TSH should also be measured to identify the rare individual not responding to brief T3 withdrawal.

Zinc metabolism in humans after oral and intravenous administration of Zn-69m.

Seventeen patients were studied after separate oral and intravenous administration of 50 muCi Zn-69m to determine if Zn-69m is suitable for studying zinc metabolism in humans and to determine if the route of administration affects kinetics. Patients stayed on a metabolic ward for each study. Activity was measured in the total body, urine, feces, blood, plasma, red blood cells, and by detectors over liver and thigh. Five day urine to fecal ratios were 0.44 (intravenous), 0.018 (oral). Most activity went rapidly to liver, then followed two component exponential loss patterns in both cases. Thigh area doubling time was 5.7 days whether the zinc was given orally or intravenously. Plasma activity decreased to less than 2% of that injected by 24 hr after intravenous administration and decreased from a maximum of 1.2% of that ingested, 3 hr after oral administration to 0.7% by 24 hr. Red blood cell activity increased through the 5-day study period to maximum values of 6.4% of that injected after intravenous administration and 2.4% of that ingested after oral administration. Similar metabolic patterns were observed regardless of whether Zn-69m was administered intravenously or orally, suggesting that these patterns were not affected by the mode of administration for the cases studied.

Absorption of orally administered 65Zn by normal human subjects.

Despite studies by several investigators of human gastrointestinal 65Zn absorption, implications of these data for evaluation of functional zinc status are unclear because limited numbers of normal subjects have been studied. To evaluated zinc absorption in normal humans, 75 subjects (31 women, 44 men, ages 18 to 84 yr) were given 10 micro Ci carrier-free 65Zn orally after an overnight fast. Absorption calculated from total body retention measured 7, 14, and 21 days after administration of tracer was 65 +/- 11% (mean +/- 1 SD), range from 40 to 86%. Comparison of these results with those for patients with a variety of diseases indicate that patients exhibit a wider range of absorption and, in four of six studies patients exhibit decreased mean zinc absorption. These results of gastrointestinal zinc absorption in a large number of normal humans offer a basis for a clearer comparison with data from patients who exhibit abnormalities of zinc absorption.

Kinky hair disease. Study of copper metabolism with use of 67Cu.

Metabolism of labeled Cu (67Cu) was studied in three patients with kinky hair disease (KHD). Labeled Cu was administered first intravenously and, later, orally. We determined oral absorption, excretion, and internal kinetics of this metal. Patients with KHD absorbed 11% to 13% of Cu given orally, compared to 46% by unaffected controls. Total excretion of Cu given intravenously during the first seven days after administration was greatly reduced in patients with KHD. The biological half-life of 67Cu in patients with KHD was increased by a factor of 2 to 3 over the normal control. Most of the labeled Cu was retained by the patient's liver, while in the control subject there was more rapid movement to the Cu to circulation (ceruloplasmin). Red blood cells of patients with KHD incorporated orally administered Cu preferentially, which was sufficient to prevent anemia.

Accidental ingestion of Tc-99m in breast milk by a 10-week-old child.

Ten weeks after the birth of her first child by Caesarian section, a 27-year-old woman was admitted for evaluation of a possible central nervous system lesion. Four hours after i.v. administration of 15 mCi of Tc-99m for a brain scan, the patient breast-fed her infant; then became concerned about the possibility of radioactivity in her breast milk. Total body gamma-ray measurements of the infant indicated ingestion of 82.5 muCi Tc-99m (corrected for effective T 1/2), while ingestion calculated from gamma-ray measurements of serial milk samples was 75.6 muCi. The single organ radiation dose for the thyroid as the critical organ was 300 mRad, whereas that to the total body was 12 mRad (values derived from 82.5 muCi determined by whole body counting). Had breast feeding been continued at 4-hr intervals, the total ingested activity would have been 94.8 muCi giving the infant a dose of 380 mRad to the thyroid and 16 mRad to the whole body. Had the feeding been given 1/2-hr after injection 726 muCi would have been ingested, increasing the dose by a factor of 10.

Acute coronary occlusion: prolonged increase in collateral flow following brief administration of nitroglycerin and methoxamine.

Regional coronary blood flow was determined with the radioactive microsphere technique 10 an 70 minutes and 2 1/2 and 5 hours after abrupt occlusion of the left anterior descending coronary artery in 12 closed chest sedated dogs. In six dogs, nitroglycerin, 200 to 400 microng/min, was infused intravenously 10 to 70 minutes after occlusion. Methoxamine was administered to return blood pressure and heart rate to prenitroglycerin levels. Ten minutes after occlusion (before treatment) collateral flow values and ischemic zone endocardial/epicardial flow ratios were equivalent in untreated (0.11+/-0.03 ml/min per g; 0.31+/-0.05) and treated dogs (0.14+/-0.02 ml/min per g; 0.29+/-0.03). In untreated dogs, collateral flow did not change over 5 hours; the endocardial/epicardial flow ratio was decreased at 5 hours (0.21+/-0.05, P less than 0.05). In contrast, in treated dogs, collateral flow and the endocardial/epicardial flow ratio were increased at 70 minutes (0.27+/-0.04 ml/min per g, P less than 0.05; 0.53+/-0.10, P less than 0.05). Most importantly, collateral flow remained elevated 5 hours after occlusion (0.26+/-0.03 ml/min per g, P less than 0.05) although treatment was discontinued 70 minutes after occlusion. Hence, collateral flow was unchanged over 5 hours of occlusion in untreated dogs, but short-term treatment with nitroglycerin and methoxamine resulted in a sustained increase in collateral flow. These findings may be a result of stimulation by nitroglycerin and methoxamine of the spontaneous rate at which intrinsic collateral function increases after ischemia. Alternatively, nitroglycerin and methoxamine may maintain cell viability until collateral vessels develop spontaneously.

Effect of long-term freezer storage, thawing, and refreezing on selected constituents of serum.

We determined the effect of long-term freezer storage and repeated thawing and freezing of serum on concentrations of electrolytes (sodium, potassium, calcium, and phosphate), enzymes (aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, and creatine kinase), total protein, tumor markers (carcinoembryonic antigen and alpha-fetoprotein), and other substances. Vials (1 ml) of frozen serum from a single blood drawing from 40 women with no breast disease and 70 with benign breast disease were analyzed annually from 1983 to 1987. Blood had been obtained from 40 subjects in 1978, 40 in 1980, and 30 in 1983. Thawing and refreezing studies were done in two ways: (1) serum samples from 30 subjects with benign breast disease were thawed at weekly intervals for 6 weeks and (2) serum samples from 30 patients with stage IV breast cancer were analyzed for alpha-fetoprotein and carcinoembryonic antigen, and serum specimens from 23 patients with benign breast disease and 7 control subjects were analyzed for lactate dehydrogenase and creatine kinase after thawing and keeping the samples at room temperature for up to 4 hours and then refreezing them. For measuring laboratory variability, duplicate samples were processed. Long-term storage (up to 10 years) and repeated thawing and refreezing did not affect the results of any tested constituents of serum. Although most measurements showed statistically significant variability over test cycles, these differences were thought to be due to laboratory variability.

A prospective randomized study of adjuvant parenteral nutrition in the treatment of advanced diffuse lymphoma: influence on survival.

The efficacy of adjuvant parenteral nutrition (PN) in cancer patients is not well defined. Twenty-one of 42 patients with advanced diffuse lymphoma were randomly selected to receive PN during an aggressive program of multiple-drug chemotherapy. These patients received 56 courses of central venous PN support during the first 14 days of the 28-day cycles of early and late phases of chemotherapy. PN patients received an average of 2,216 kcal/day, and their oral intake was 836 kcal/day during therapy. PN patients had marked weight gains, and standard nutrition (SN) patients had stable weights both during cycles of therapy and over the entire course of therapy. Lean body mass, as indicated by total body potassium, anthropomorphic measurements, serum albumin, creatinine-to-height ratio, total iron-binding capacity, and total lymphocyte count, was not improved in PN patients as compared to SN patients. PN therapy was complicated by symptomatic subclavian vein thromboses, septic catheters, and pneumothoraxes. The actuarial survival rate at 2 years was 68.8% +/- 10.9% (estimate +/- SD) for PN patients and 65.8% +/- 12.2% (estimate +/- SD) for SN patients. These results suggest that the weight gained by PN patients was composed of fat, water, or both. A lack of improvement in nutritional status and survival rates and a significant complication rate were associated with adjuvant PN during chemotherapy.

Zinc metabolism in adrenal cortical insufficiency: effects of carbohydrate-active steroids.

Detailed studies of zinc kinetics were performed in two patients with adrenal cortical insufficiency to investigate the effects of carbohydrate-active steroids (CAS) on zinc metabolism. Zinc- 69m was administered intravenously to each patient under two conditions: (1) treated with CAS replacement therapy and (2) untreated, ie, without hormone treatment for five to six days. Radioactivity was measured in blood plasma, red blood cells, urine, and stool and by means of external probes placed over liver and thigh. Data were analyzed using a previously developed multicompartmental model, which describes the early phase of zinc metabolism. The results of these studies suggest that CAS promotes the internalization of zinc into red blood cells and liver cells. These results are consistent with previous in vitro and in vivo studies in which CAS was shown to induce the synthesis of metallothionein in liver cells.

Effects of oral zinc loading on zinc metabolism in humans--I: Experimental studies.

The effects of oral zinc on distribution, retention and excretion of orally administered 65Zn were studied in 50 patients with taste and smell dysfunction. The study was conducted in three phases. In the first phase all patients were studied for 21 days after receiving 3-18 microCi of 65Zn as ZnCl2 orally after an overnight fast. In the second phase, started after 21 days and continued for 290 to 440 (mean 336) days, all 50 patients received placebo for ZnSO4. In the third phase 14 patients continued on placebo while 36 received ZnSO4 (100 mg/day Zn++) for 112 to 440 (mean 307) days. Phases two and three were a controlled clinical trial of the effects of zinc on retention of 65Zn tracer. Total body retention and activity in plasma and red cells were measured for all patients throughout the study. Ten of the 36 patients treated with ZnSO4 had additional measurements of 65Zn activity in liver and thigh made using external detectors. Total body retention during the second phase placebo period was not significantly different (p greater than 0.25) for the 36 subjects subsequently treated with ZnSO4 (biological half-time (Tb) 378 +/- 12 days) (mean +/- SEM) and the 14 who were continued on placebo through the third phase of the study (Tb = 384 +/- 8 days). During the third phase patients receiving ZnSO4 showed an accelerated loss of total body 65Zn (Tb = 235 +/- 8 days) which was significantly different (p greater than 0.001) from half-time values during placebo treatment. Accelerated loss of 65Zn from the thigh was apparent immediately while that from the liver began after a mean delay of 107 days. There was no apparent effect of zinc on loss of mean 65Zn activity from red blood cells.

Effects of oral zinc loading on zinc metabolism in humans II: in vivo kinetics.

The effects of oral zinc loading on zinc metabolism were studied in 10 patients with taste and smell dysfunction following oral administration of Zn-65 (physical t1/2 = 245 d) and subsequent administration of oral stable zinc. Patients took an ad libitum dietary zinc intake of 8-13 mg daily for 290-440 days (mean, 336) following Zn-65 administration, followed by an intake of an additional 100 mg/day of zinc ion (as ZnSO4) over the next 112-440 days (mean, 307). A previously developed compartmental model, based on five day studies of patients with taste and smell dysfunction, was extended in such a way that it was consistent with both short term and long term kinetics. In this extended model, the turnover of 90% of total body zinc, previously unaccounted for by the kinetics in the short term studies could be explained by a single compartment, as postulated in the short term studies. Using the model, it was found that changes in the rate constants for gastrointestinal absorption and renal excretion of zinc were both necessary and sufficient to explain the changes seen in the kinetic curves following oral zinc loading. Michaelis-Menten type saturation mechanisms were adequate to explain the observed parameter changes. These changes also accounted for the observed mean plasma zinc mass increase of only 37% above pre-load levels in face of an 11-fold increase in zinc intake.

Body cell mass in cancer-bearing and anorexic patients.

We used a sensitive whole body counter which measures potassium-40 (40K) to determine total body potassium and to estimate body cell mass (BCM) in 104 previously untreated patients with upper gastrointestinal malignancies, 233 normal volunteers, and 18 patients with anorexia nervosa. BCM was greater in normal males than in females. In both normal males and females, the BCM tended to decrease with age, both as an absolute measure and as a percentage of body weight. Anorexia nervosa patients experienced marked weight loss (30.5%), and had significant depletion of absolute BCM, but exhibited relative sparing of BCM as indicated by a rise in BCM as a percentage of body weight. This may reflect a normal adaptation and predominant fat utilization in chronic malnutrition. The cancer patients, on the other hand, had significant weight loss (12.7% for females, 13.9% for males) and demonstrated a proportional decline in BCM, with no change in BCM as a percentage of body weight. These findings support the contention that, in the cancer-bearing patient, weight loss consists of a significant depletion of both fat and BCM. The challenge to the clinicians caring for cancer patients is repletion of this supremely functional body compartment.

Providing human tissues for research: how to establish a program.

The Cooperative Human Tissue Network is a group composed of cooperating academic institutions that supply human tissues to researchers studying a wide range of neoplastic and other diseases. The experience of the Cooperative Human Tissue Network in establishing methods of prospective tissue collection and in developing tumor banks is discussed to aid institutions in establishing tissue resources for their local investigators, who may wish to use human tissues in current or future research projects. The advantages to pathology departments and to associated medical institutions of establishing an organized tissue resource include ensuring proper institutional review board approval of research projects using human tissues, protecting diagnostic specimens, creating new opportunities for extramural research, increasing the speed of diagnostic specimen transport to surgical pathology, and providing educational and research opportunities for pathologists and pathology residents. Methods of tissue collection, processing, storage, data collection, and supply are outlined. Also, resources necessary to begin organized tissue collection, including personnel, space, equipment, and supplies, are discussed.

External gamma-ray counting of selected tissues from a Thorotrast patient.

Results of gamma-ray measurements of selected tissues from a patient who was injected with Thorotrast almost 36 y ago are reported. The purposes of this study were: 1) to determine the relative tissue distribution and activities of specific radionuclides in the 232Th decay chain, specifically 228Ra (as measured by 228Ac), 212Pb, and 224Ra (measured directly and as measured by 212Pb), and 2) to evaluate the level of radioactive disequilibrium among the daughter products. The spleen and liver had the highest concentrations of radioactivity. Bone also appears to be a long-term sink for 232Th daughter products based on estimates from a small portion of one rib. Larynx and esophagus contained measurable activity, which may have been due to their proximity to the "Thorotrastoma." Radioactivity in the remaining measured tissues were low, as expected. Secular equilibrium could be demonstrated in bone, pancreas, larynx, esophagus, and breast. Significant disequilibrium was observed for spleen, liver, kidney, and red blood cells. Radioactivity measurements reported here will be useful in estimating radiation doses to selected tissues. Such dose estimates are valuable in refining current risk estimates (e.g., liver) and in identifying tissues at risk for further epidemiologic studies. These results, while consistent with other published studies, should be interpreted with caution since measurements were made on only one patient.