Search

Recent discoveries in cancer biology have greatly increased the understanding of cancer at the molecular level, but translating this knowledge into clinically useful diagnostic tests has proved challenging. More efficient transfer of new molecular tests into patient care requires better standardization of laboratory practices, measurement methods and data management. The workshop assembled experts from National Cancer Institute, US FDA, National Institute of Standards and Technology, academia and industry, to address the most efficient approaches to biomarker standardization and validation. The workshop participants described the current state of research in molecular diagnostics standardization and addressed three questions: what has worked? What has not?And what needs improving?

A delayed onset of diabetes is characteristic of subtotally pancreatectomized patients in whom persistent hyperglycemia per se is documented to lead to the development of insulin resistance. This study was conducted to elucidate the metabolic alterations seen during transition of the acute to chronic phase after subtotal pancreatectomy (SP). Eight male Sprague-Dawley rats were studied 2 weeks after surgery in the acute phase, and the other eight at 4 weeks in the chronic phase. Phosphoenolpyruvate carboxykinase (PEPck) for gluconeogenesis and the malic enzyme for de novo fatty acid synthesis in the liver showed a reciprocal change, the former activity being increased, while the latter was suppressed. Both alterations were more pronounced in the chronic phase. In the acute phase, lipoprotein lipase (LPL) for triglyceride clearance decreased in the adipose tissue, while that in the cardiac and skeletal muscle became significantly elevated. The latter elevations were decreased in the chronic phase. Sustained hyperglycemia in the SP rats not only increased the changes in PEPck and malic enzyme activities but reversed the tissue-specific muscle LPL elevations. These changes might help to explain the wasting condition seen in surgically induced diabetic patients.

To better understand the role of the liver in the hypertriglyceridemia observed in a tumor-bearing state, we have examined tumor-induced alterations in hepatic lipogenesis and fatty acid oxidation. The effects of differing tumor burden as well as tumor excision on the activity and mRNA levels of malic enzyme and carnitine palmitoyltransferase were studied in Fisher 344 rats bearing a methylcholanthrene-induced sarcoma. Serum triacylglycerols and plasma nonesterified fatty acids (NEFA) levels were both elevated with increasing tumor burden (P < 0.05 vs control). The elevation disappeared with tumor removal. Malic enzyme activity of tumor bearers, compared with control rats, declined with an increase in tumor burden. These two variables were negatively correlated (r = -0.90, P < 0.01). The changes in activity were accompanied by positively correlated changes in mRNA levels (r = 0.73, P < 0.01). Carnitine palmitoyltransferase activity was not altered, even in the presence of a large tumor burden. Hepatic lipogenesis, reflected by malic enzyme activity, was tumor-dependent and was significantly reduced during the period of circulating hypertriglyceridemia. Fatty acid oxidation, reflected by carnitine palmitoyltransferase activity, was not enhanced in spite of an ample supply of NEFAs to the liver from the peripheral tissues. The data suggest that neither hepatic lipogenesis nor fatty acid oxidation contribute to hypertriglyceridemia in the tumor-bearing state.

Although blood flow is central to systemic metabolism, little is known about the effect of tumor on the perfusion of host tissues. This study evaluated the effects of a methylcholanthrene-induced sarcoma on blood flow to intra-abdominal organs and skeletal muscle of Fischer-344 rats anesthetized with pentobarbital sodium. Animals were studied by aortic injection of radiolabeled microspheres when the tumors reached 20% of body weight. Total-organ arterial flows in spleen, liver, small intestine, and pancreas were each increased to 50-150% in tumor bearers relative to controls (P less than 0.05). Portal venous flow and flow per gram to hindlimb muscle were 60 +/- 20 and 300 +/- 100% greater, respectively, in tumor-bearing animals (P less than 0.005). This study shows that tumor growth can be associated with large changes in organ flow and distribution of cardiac output. The increase in skeletal muscle flow in the tumor bearers, which lost normal tissue weight relative to pair-fed controls (P less than 0.05), is in marked contrast to decreased muscle flow previously observed in simple starvation.

With the euglycemic clamp technique, we evaluated the effects of graded doses of insulin on glucose turnover rates and forearm lactate balance in five weight-losing patients with cancer before surgery and five age- and weight-matched healthy volunteers (control subjects). Insulin was infused sequentially at increasing rates of 0.5 (low physiologic), 1.0 (high physiologic), and 4.0 (supraphysiologic) mU/kg.min for 120 minutes each. Concurrently, rates of glucose appearance and disappearance were derived from [3-3H] glucose infusion. The mean postabsorptive rate of glucose appearance in patients (2.9 +/- 0.1 mg/kg.min) was significantly higher (p less than 0.02) than that of control subjects (1.98 +/- 0.16 mg/kg.min). Complete suppression of endogenous glucose production occurred at high physiologic insulin concentrations. With progressive insulin infusion, the rate of glucose disappearance increased to 3.6 +/- 1.2, 8.7 +/- 0.8, and 13.7 +/- 1.1 mg/kg/min in control subjects and 2.9 +/- 0.4, 5.3 +/- 0.3, and 10.9 +/- 0.9 mg/kg.min in patients, significantly different from that of control subjects (p less than 0.05) during the intermediate (high physiologic) insulin infusion. A comparable slight increase in arterial plasma lactate concentration was observed in both groups with progressive hyperinsulinemia. Baseline peripheral lactate flux was identical in patients (-272 +/- 56 nmol/100 gm.min) and in controls (-271 +/- 57 nmol/100 gm.min). Progressive physiologic hyperinsulinemia resulted in significantly (p less than 0.05) augmented peripheral lactate efflux in patients (-824 +/- 181 nmol/100 gm.min) compared with control subjects (-287 +/- 64 nmol/100 gm.min). Supraphysiologic insulin abolished this increased lactate efflux in patients. Postabsorptive rates of endogenous glucose appearance in weight-losing patients with cancer were elevated, but complete suppression was achieved with insulin concentrations in the physiologic range. Total body glucose use was diminished in these patients, consistent with a state of insulin resistance. This impaired insulin action on peripheral glucose use was associated with an increase in peripheral lactate release in patients.

Pretransplant blood transfusion has been shown to significantly affect the outcome of renal transplantation. Evidence regarding the association of blood transfusions with growth or recurrence of solid tumors is still conflicting both in clinical and in experimental studies, although diminished survival has been suggested in several studies. To determine the influence of blood transfusions and hypovolemia, as separate or combined factors, on tumor growth, we evaluated the weight of a subcutaneously implanted sarcoma (methylcholanthrene-induced) in 35 rats. After reaching 1% tumor burden (day 0), the animals were separated into two groups: hypovolemia (shed volume, 15 ml/kg) or normovolemia. These groups were further divided according to resuscitation: OO (no resuscitation), BL (receiving syngeneic blood stored in citrate phosphate dextrose for 4 days, 15 ml/kg), SL (receiving 0.9% sodium chloride, 45 ml/kg). Tumor dimensions were determined daily by external measurement, and tumor weight was calculated. Hypovolemia exerted a significant influence on tumor growth, independent of the resuscitation modality. The rats that received blood transfusions showed an increased rate of tumor growth, compared to the animals that received saline solution or no treatment. No interaction was noted between the effects produced by hypovolemia and blood transfusion. We conclude that the hypovolemic event enhanced tumor growth independently of the resuscitation, and transfusion of citrate phosphate dextrose-blood stored for 4 days did influence tumor growth in this model. We suggest that the effect of blood transfusion in patients with cancer has to be redefined to account for the influence of possible hypovolemic events.

To elucidate the mechanisms of hypertriglyceridemia observed in the tumor-bearing rat, tissue lipoprotein lipase (LPL) activity and LPL mRNA levels were examined in the fed and fasted states at different degrees of tumor burden and after tumor removal. LPL activity in the epididymal fat pad and cardiac muscle in the 24-h-fasted rats was significantly decreased with increasing tumor burden (r = -0.53, P less than 0.05 and r = -0.72, P less than 0.01, respectively). Tumor removal completely reversed these changes. In contrast, no change in LPL activity was detected in the fed state since food intake stimulated LPL activity to the same extent in both tumor-bearing (TBR) and control rats. LPL activity in the diaphragm and skeletal muscle was only marginally altered in TBR, as compared to controls. LPL mRNA from the epididymal fat pad and cardiac muscle migrated to the same site on agarose gel and hybridized to a LPL-specific complementary DNA probe. The decline in LPL activity in epididymal fat pad observed in TBR was associated with a decrease in LPL mRNA levels. In contrast, there was no significant difference in LPL mRNA levels in cardiac muscle between the two groups despite significantly suppressed enzyme activity in tumor bearers. This study provides evidence that hypertriglyceridemia in TBR is due in part to tumor-dependent suppression of adipose and cardiac LPL activity in the fasted state, which is stimulated by the presence of tumor. Unlike cardiac LPL, the tumor-induced changes in adipose LPL activity are regulated at the mRNA level in this tumor model.

We examined the effect of prolonged bile duct obstruction, and subsequent biliary decompression, on biochemical and metabolic parameters, using a reversible jaundice model in male Fischer 344 rats. The animals were studied after biliary obstruction for varying periods (4 days, one week, and two weeks) and following decompression. They were sacrificed one or two weeks following decompression. All the rats were compared to sham operated, pair-fed, controls. Obstructive jaundice rapidly increased bilirubin, liver enzymes, serum free fatty acid, and triglyceride levels. Glucose levels were significantly decreased in the jaundice rats compared to their pair-fed controls. Only after two weeks of jaundice was significant hypoalbuminemia observed. Following decompression, all biochemical and metabolic values gradually returned to normal levels, except for albumin. Hypoalbuminemia was not reversed within the two-week post-decompression period. The rats jaundiced for two weeks had significantly higher mortality, compared to the other groups. We conclude that prolonged jaundice adversely affects the metabolic capacity of the rats, with albumin concentration being markedly decreased, and that biliary decompression could not reverse completely all the alterations seen with cholestasis, especially following two weeks of bile duct obstruction.

Cancer cachexia is frequently accompanied by hyperlipidemia. To identify the mechanisms underlying this alteration in lipid metabolism, free fatty acid (FFA) and very low density lipoprotein-associated triacylglycerol (VLDL-TG) kinetics were determined in tumor-bearing (subcutaneously implanted methylcholanthrene-induced sarcoma) Fischer 344 rats. The animals were studied after chronic vascular catheterization, in an unanesthetized, undisturbed state, after 24 hr of fasting. They were separated into three groups: control (n = 5), tumor-bearing (n = 5, tumor burden = 10% body weight), and 7 days after tumor excision (n = 5). VLDL-TG and FFA kinetics were assessed by a constant infusion of [3H]palmitate-labeled VLDL-TG and [14C]palmitate bound to albumin, respectively. FFA rate of appearance (FFA-Ra) and clearance (FFA-Cl) and VLDL-TG rate of appearance (VLDL-TG-Ra) and clearance (VLDL-TG-Cl) were determined at steady state. We observed that the tumor-bearing rats had significantly increased FFA-Ra and VLDL-TG-Ra, decreased VLDL-CL, and maintained FFA-Cl, when compared to control. These alterations returned to normal levels after tumor excision. The results suggest that the hyperlipidemia observed in tumor-bearing rats is due to elevated lipolysis rate, maintained rate of plasma FFA clearance, increased triacylglycerol production and VLDL secretion by the liver, and decreased VLDL-TG clearance from plasma. These alterations were reversed 7 days following tumor excision.

A model of reversible, extrahepatic biliary obstruction is described. Vessel loop blockade of the biliary tree results in obstructive jaundice while removal of the exteriorized vessel loop provides internal biliary drainage without subsequent laparotomy. This technique combined with a system for chronic venous infusion and arterial blood sampling in the unrestrained rat is ideal for long-term metabolic studies of obstructive jaundice. Male Fisher 344 rats (275-350 g) underwent either the combined procedure of total biliary tract blockade and vascular access or sham operation. Mean serum bilirubin was significantly elevated (12.7 +/- 8.9 mg/dl) in the experimental group and following relief of biliary obstruction significantly dropped below 1 mg/dl in all animals except one. Concomitant changes in alkaline phosphatase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase were seen. Experimental and control rats initially lost weight following laparotomy; however, mean body weight stabilized by the 5th postoperative day and was similar in both groups on the 10th postoperative day. This combined procedure is a simple, effective and reproducible method of obstructive jaundice.

The plasma-free fatty acid response to intravenous glycerol infused at 250 and 500 mumol/min was determined in five normal volunteers in the postabsorptive state. There was a drop in free fatty acid concentration in all five subjects (one-way ANOVA, p less than 0.01) after the glycerol infusion with no change in insulin concentration compared to the post-absorptive state. These results suggest that intravenous glycerol infusions decrease free fatty acid concentrations in the post-absorptive state by an insulin-independent mechanism. When pharmacologic nonisotopic glycerol infusions are used to determined lipolytic rate, simultaneous measurement of free fatty acid concentrations should be interpreted with caution.

The accelerated mobilization of peripheral protein and subsequent increased gluconeogenesis are regarded as mechanisms of cancer cachexia. To determine the relation of gluconeogenesis to different degrees of tumor burden and subsequent tumor removal in the fed and fasted states, we examined the activity and mRNA levels of the key regulatory enzyme for gluconeogenesis: phosphoenolpyruvate carboxykinase (PEPck) in the liver of Fischer rats with a transplanted methylcholanthrene-induced sarcoma. PEPck activity in liver cytosol, after a 24-hour fast, was significantly higher in the tumor-bearing rats than in their pair-fed controls. The increase in enzyme activity was clearly evident at 8% tumor burden and correlated positively with the degree of tumor burden (r = 0.85, p less than 0.01). Removal of the tumor produced a complete reversal of PEPck activity 10 days after excision. Regular feeding also abolished this increased enzyme activity. A similar trend was seen in the mitochondria. PEPck mRNA levels of rats with greater than 11% tumor burden in the fed state were decreased more than those of controls. PEPck mRNA levels were equally elevated in tumor bearers and controls in the 24-hour-fasted state. These results suggest that tumor-bearing simulates the fasted state associated with hypoglycemia, which in turn triggers induction of the gluconeogenic enzyme, PEPck.

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.

A PTH-related peptide (PTHRP) has been identified and its cDNA cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The human PTHRP gene has been recently isolated and found to be a complex transcriptional unit using multiple promoters and containing alternatively spliced 3' exons which result in three mRNA classes, each class encoding a PTHRP with a unique carboxy-terminus. The PTHRP gene appears to be expressed in a number of normal tissues, and PTHRP transcripts have been previously reported to be overexpressed in a small sample of human parathyroid adenomas. In the present study we surveyed RNA prepared from a total of 60 abnormal human parathyroid glands for PTHRP gene expression using a combination of Northern blotting and RNase protection techniques. Apparent overexpression of PTHRP mRNA was observed in two thirds of parathyroid adenomas, whereas no overexpression was found in 7 examples of sporadic primary hyperplasia, 5 examples of secondary hyperplasia, and 3 examples of parathyroid carcinoma. Apparent overexpression was also observed in 1 of 4 cases of multiple endocrine neoplasia type 1, 1 of 2 examples of multiple endocrine neoplasia type 2, and 1 gland considered to represent tertiary hyperparathyroidism. Northern analysis of poly(A)+ RNA prepared from three representative adenomas using region-specific probes indicated that two putative promoters are used and revealed a pattern of preferential splicing of transcripts to include the most distal 3' exon. These findings suggest that the PTHRP gene is commonly overexpressed in adenomatous parathyroid glands, but not in sporadic primary hyperplasia, that this overexpression does not seem to be dependent on the use of a single specific promoter, and that adenomatous parathyroid cells appear to preferentially use one of several alternative splicing pathways. It is presently not known whether PTHRP is secreted by abnormal parathyroid tissues and, if so, in what form.