Enterostatin inhibition of angiogenesis: possible role of pAMPK and vascular endothelial growth factor A (VEGF-A).

INTRODUCTION: We utilized a genomic analysis of the response of neuronal GT1-7 cells to enterostatin to identify pathways responsive to this peptide. This information, together with reported properties of the enterostatin receptor, suggested that enterostatin may have an effect on angiogenesis. METHOD: To investigate this hypothesis, we studied the effect of enterostatin as an antiangiogenic agent in two angiogenic tissue culture model systems. RESULTS: Enterostatin induced a 50% or greater inhibition in the angiogenic response of human fat cells and had a U-shaped bimodal dose-response effect in inhibiting angiogenesis in a human placental vein angiogenesis model. To further understand this response, we tested enterostatin's effect in a human hepatoma cell line (HepG2 cells) that was subjected to glucose deprivation, a condition known to induce angiogenesis in other tumor cell lines. Phosphorylated AMP kinase (pAMPK) levels and vascular endothelial growth factor A (VEGF-A) mRNA expression were elevated robustly after incubation of HepG2 cells in the absence of glucose for 4 h, but 15 min incubation with enterostatin dramatically inhibited this pAMPK activation and reduced VEGF-A gene expression after 1 h incubation with enterostatin. The AMPK activator 5-aminoimidazole-4-carboximide ribonucleoside (AICAR) induced VEGF-A expression. SUMMARY: These data suggest that enterostatin has an antiangiogenic effect and suggest that it regulates VEGF-A gene expression through inhibition of AMPK activity.

Characterization of the in vivo and in vitro inhibition of gastrin secretion from gastrinoma by a somatostatin analogue (SMS 201-995).

Earlier experiments characterized the effect of SMS 201-995 on gastrin secretion from gastrinoma in vivo. The results showed that the somatostatin analogue inhibits basal as well as secretin- and calcium-stimulated gastrin secretion. The current study examined the effect of SMS 201-995 on gastrin secretion from gastrinoma in vitro. Gastrinoma cells were prepared in cell culture or acute cell dispersion to study basal gastrin release. In cell culture, SMS 201-995 at 10(-9) M, 10(-8) M, and 10(-7) M significantly stimulated gastrin secretion (basal medium gastrin, 157 +/- 7.9 pg/ml; with SMS 201-995 10(-9) M, 786 +/- 62 pg/ml; with SMS 201-995 10(-8) M, 569 +/- 72 pg/ml; and with SMS 201-995 10(-7) M, 258 +/- 26 pg/ml). In contrast, in acute cell dispersion, the somatostatin analogue inhibited gastrin secretion (basal medium gastrin, 12.8 +/- 1.3 ng/ml; with SMS 201-995 10(-9) M, 9.0 +/- 0.1 ng/ml; with SMS 201-995 10(-8) M, 8.4 +/- 1.5 ng/ml; and with SMS 201-995 10(-7) M, 7.9 +/- 0.2 ng/ml). Gastrinoma cells were prepared in cell culture to study the effect of SMS 201-995 on gastrin secretion stimulated by secretin and by post-receptor increases in adenosine cyclic nucleotide. The somatostatin analogue inhibited gastrin secretion stimulated by secretin (10(-6) M) (797 +/- 48 pg/ml for secretin alone, compared with 396 +/- 9.4 pg/ml for secretin plus SMS 201-995). SMS 201-995 did not inhibit gastrin secretion stimulated by dibutyryl cyclic AMP (10(-7) M) (617 +/- 62 pg/ml for dibutyryl cyclic AMP alone, compared with 778 +/- 55 pg/ml for the two together). In vitro, SMS 201-995 inhibits basal gastrin secretion from gastrinoma prepared in acute cell dispersion, but not gastrinoma in cell culture, probably due to differences in basal secretory rates. The effect in vitro is less than that in vivo. SMS 201-995 does not inhibit postreceptor increases in adenosine nucleotide. This indirectly supports the hypothesis that SMS 201-995 acts in gastrinoma cells to inhibit gastrin secretion by inhibition of adenylate cyclase activity.

Role of peptide radioimmunoassay in understanding peptide-peptide interactions and clinical expression of gastroenteropancreatic endocrine tumors.

Peptide radioimmunoassay has become an important clinical and research tool in understanding the role of peptides in the pathophysiology of gut endocrine tumor syndromes. A gut peptide radioimmunoassay laboratory has been established for the diagnosis and clinical monitoring of endocrine tumors of the gastroenteropancreatic (GEP) system. Radioimmunoassay has enhanced our awareness that co-occurring peptide interactions may modify and ultimately influence the clinical expression of these tumors. Furthermore, it has helped develop a rationale for the use of prototype peptides such as somatostatin and its long-acting analogue Sandostatin (SMS 201-995) in the management of GEP tumors. This group's experience, as well as the experience of other investigators, is presented, and the clinical utility of peptide radioimmunoassay in the field of gut endocrinology is demonstrated.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Acute gastric pH changes alter intraluminal but not plasma peptide levels.

Gastric acidity is influenced by systemic and local peptide effects. Previous work by others has shown that intraluminally secreted peptides may have a role in local control of gastric acidity; however, the response of these peptides to acute changes in gastric pH is unknown. To determine the effects of acute changes in pH on systemic and intraluminal peptide levels, 14 normal volunteers underwent placement of a nasogastric tube after an overnight fast. Blood and gastric fluid were analyzed on a control day, 2 hours after completion of 24 hours of aluminum-magnesium antacid therapy and after 24 hours of H2 blockade. Plasma and acid-alcohol-extracted gastric peptide levels were measured with specific radioimmunoassays. Specimens were subdivided into two groups: 28 gastric fluid specimens with a pH less than 4 and 10 specimens with a pH greater than 4. In the patients with a pH greater than 4, the luminal peptides, motilin, neurotensin, pancreatic polypeptide, somatostatin, substance P, and gastrin, were decreased by 50% to 90% and gastrin-releasing peptide was decreased by 36% compared with specimens with a pH less than 4. Conversely, intraluminal vasoactive intestinal polypeptide and calcitonin levels were elevated by 60% and 27%, respectively, in the samples with a pH greater than 4. Intraluminal peptide concentrations are responsive to changes in intragastric pH; however, this response was not seen in plasma peptide levels.

Comparison of adrenal and extraadrenal pheochromocytomas.

BACKGROUND: It is commonly believed that extraadrenal tumors (EAT) of the paraganglion system are more likely to be malignant than adrenal tumors (AT) and carry a poorer prognosis. We analyzed 73 paraganglion tumors (PT) to determine whether EATs are more likely to be malignant or have a poorer prognosis than ATs. METHODS: A review of patients with PTs at three institutions was performed. Malignant tumors were defined as those that metastasized. Comparison of the frequencies of malignant tumors was performed by chi-squared analysis. Survival distributions were determined by Kaplan and Meier analysis. Comparison of survival distributions was performed by log-rank analysis. RESULTS: There were 73 patients. There were 51 ATs, of which 24 were malignant, and 22 EATs, of which 11 were malignant (p = 0.82). The 5-year survival rate was 77% for patients with ATs and 82% for patients with EATs (p = 0.29). The 5-year survival rate for patients with malignant ATs was 57%, and 74% for patients with malignant EATs (p = 0.15). There were no significant differences in disease-free survival rates on the basis of tumor site. CONCLUSIONS: We were unable to demonstrate that EATs are significantly more likely to be malignant than ATs. The survival and disease-free survival rates for malignant ATs and EATs are similar, and among malignant tumors, there may be no prognostic value of the anatomic location.

Postreceptor signal transduction mechanisms involved in octreotide-induced inhibition of angiogenesis.

BACKGROUND: Somatostatin analogues inhibit peptide release and cell growth through multiple postreceptor signal transduction mechanisms (PRSTM), including G proteins (GP), cyclic adenosine monophosphate (cAMP), calcium, protein kinase C (PKC), and tyrosine phosphatase (TP). Octreotide acetate (OA), a somatostatin analogue, has been shown to inhibit angiogenesis; however, the PRSTM involved are unknown. METHODS: Fertilized chicken eggs were obtained and incubated. On day 3, embryos were removed and placed in plastic wrap hammocks. On day 7, disks containing OA, test substances that interfere with PRSTM, or combinations of OA plus a test substance were placed on the developing chorioallantoic membrane. Blood vessel growth under each disk was assessed at 24 hours. Data were evaluated by chi-squared analysis. RESULTS: OA's ability to inhibit angiogenesis is significantly diminished when combined with calcium, bradykinin (increases calcium), pertussis toxin (inhibits GP), or 3-isobutyl-1-methylxanthine (increases cAMP). In contrast, no significant decrease is noted in OA's ability to inhibit angiogenesis when combined with phorbol ester (activates PKC) or vanadate (inhibits TP). CONCLUSIONS: OA-induced inhibition of angiogenesis is GP, calcium, and cAMP dependent and is PKC and TP independent. Better understanding of the PRSTM involved with OA-induced inhibition of angiogenesis may lead to enhancement of OA's effect on angiogenesis.

ABO(H) cell surface antigens in parathyroid adenoma and hyperplasia.

Forty-three cases of primary hyperparathyroidism were studied with the specific red cell adherence test (SRCA) to determine the presence or absence of ABO(H) cell surface antigens on abnormal parathyroid tissue. Of the 27 patients with the clinicopathologic diagnosis (CPD) of adenoma, 24 (89%) had lost the ABO(H) cell surface antigen of the abnormal gland. Among the 15 patients with the CPD of hyperplasia, the parathyroid tissue from three (20%) had lost its red cell antigen. In one patient, a metastasis from a parathyroid carcinoma had lost the ABO surface antigen. Several patients in whom conflicting SRCA and CPD were obtained had factors that raised doubts as to the validity of their CPD. The SRCA is a simple test that may aid in the difficult differentiation between parathyroid adenoma and hyperplasia.

Observations on the effect of a somatostatin analog in the Zollinger-Ellison syndrome: implications for the treatment of apudomas.

Somatostatin is known to inhibit hormone release and gastrointestinal secretion and hence may be useful in the treatment of amine precursor uptake, decarboxylase tumors. Clinical application has been limited by the short half-life, potency, and specificity of the natural hormone. Our study evaluated the effect of a synthetic analog of somatostatin, SMS 201-995 (Sandoz, Inc., E. Hanover, N.J.) on basal and stimulated gastrin release and gastric acid secretion in 10 patients with the Zollinger-Ellison syndrome. In experiment 1, H2-receptor antagonists were discontinued for 48 hours; SMS 201-995, 1 microgram/kg, was given subcutaneously; gastrin and SMS levels in plasma were determined by radioimmunoassay; and gastric secretion was measured and titrated at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, and 18 hours. The mean +/- SEM baseline gastrin level (1526 +/- 733 pg/ml) was significantly inhibited for 16 hours (p less than 0.05, paired t test). Gastric secretion was neutralized for as long as 18 hours (p 0.05). In experiment 2, three patients received either a secretin (2 U/kg) or a calcium stimulation test (2 mg/kg) with or without pretreatment with SMS 201-995, 1 microgram/kg, subcutaneously. The mean +/- SEM interpreted change in gastrin (ng X 60 min/ml) without SMS 201-995, 36.8 +/- 11 (secretin), and 129 +/- 30 (calcium) were reduced with SMS 201-995 to -1.1 +/- 0.76 (secretin) and -29 +/- 28 (calcium) (p less than 0.05). In the Zollinger-Ellison syndrome, SMS 201-995 caused significant and long-lasting inhibition of both tumor gastrin release and gastric acid secretion, probably by direct action on both the gastrinoma and the stomach. SMS 201-995 blocks acid secretion and secretin- and calcium-stimulated gastrin release, indicating that SMS 201-995 inhibits peptide secretion by postreceptor mechanisms. SMS 201-995 will be useful in the palliative treatment of apudomas.

Detection of occult gastrinomas with iodine 125-labeled lanreotide and intraoperative gamma detection.

BACKGROUND: Surgical exploration for gastrinoma has a high failure rate because of small primary tumors and occult metastasis. Despite extensive preoperative and intraoperative tumor localization, only 30% to 40% of patients with gastrinoma are cured by surgery. Two patients with unlocalized gastrinomas were studied with intraoperative gamma detection of an iodine 125-labeled somatostatin analog, lanreotide, to localize their tumors. METHODS: Both patients were challenged before operation with 100 micrograms of octreotide acetate, and both had circulating gastrin levels suppressed by greater than 50%. Iodine 125-labeled lanreotide (100 to 150 muCi) was injected during exploration, and an intraoperative gamma detector was used to detect tumor binding of the analog. RESULTS: In patient 1 a single source of increased counts was discovered in a retroduodenal lymph node, which was excised; no other tissue was removed. Histologic study of this node failed to demonstrate tumor; however, the patient's gastrin level was normal (63 pg/ml) 4 months after operation. In patient 2 five areas of increased counts were discovered and excised. Three of these five areas had visible tumor on microscopic examination. Three months after operation the patient's fasting gastrin level was 103 pg/ml. CONCLUSIONS: Intraoperative gamma detection of radiolabeled peptides may allow the localization of occult tumors that contain specific peptide receptors.

Effect of secretin on gastrin release from gastrinoma cells in vitro.

Secretin appears to have a direct effect on gastrinoma cells, which results in a paradoxic increase in the serum gastrin level in patients with the Zollinger-Ellison syndrome. To evaluate this effect, gastrinoma cells were challenged with secretin in acute cell dispersion or after 2 weeks in culture. Acutely dispersed cells were incubated for 15 minutes or 3 hours with and without secretin 10(-6) mol/L. (sec). There was no significant difference in gastrin release between control and sec groups in the two acute incubation periods. At 15 minutes the control value was 47 +/- 1 and the sec value was 54 +/- 1 ng/ml, while at 3 hours the control value was 59 +/- 1 and the sec value was 61 +/- 1 ng/ml. In the cell culture preparation, secretin stimulated gastrin release at all doses (control 463 +/- 85, SEC 10(-10) mol/L 603 +/- 37, sec 10(-8) mol/L 706 +/- 37, sec 10(-6) mol/L 798 +/- 48 pg/ml ([p less than 0.05, Student t test]). These results indicate that secretin stimulates gastrin release directly from cultured gastrinoma cells in a dose-dependent manner but does not stimulate gastrin release from acute cell dispersion. This might be attributed partly to the recovery of secretin receptors in the cultured cells.

Breast cancer increases initiation of angiogenesis without accelerating neovessel growth rate.

BACKGROUND: Recurrence and mortality rates in patients with breast cancer correlate with the degree of tumor angiogenesis (angiogenic index). We have developed a novel angiogenesis model by using disks of fresh human placental vein that initiate an angiogenic response and exhibit linear radial capillary growth in culture. We hypothesized that the addition of human breast cancer cells to this human placental vein angiogenesis model would increase the incidence of angiogenesis and accelerate the rate of neovessel growth compared with vein disk cultured without tumor cells. METHODS: To test this hypothesis, vein explants from seven human placentas were incorporated into clots of 0.3% fibrin in Medium 199 and fetal bovine serum with or without 1.5 x 10(5) T-47D (n = 6 placentas) or MCF-7 (n = 1 placenta) breast cancer cells. Statistical differences between the experimental (with breast cancer cells) and control (no added cells) cultures were determined by repeated measures ANOVA. RESULTS: The proportion of disks exhibiting neovessel growth (initiation) by day 12 was significantly increased in the presence of T-47D cells (p < 0.05 at day 12, p < 0.001 at day 15). No statistical difference was seen in rates of neovessel growth (millimeters per day). Similar results were seen with MCF-7 cells. CONCLUSIONS: Tumor enhancement of angiogenesis may occur by increased initiation of the angiogenic response. Subsequent vessel growth rates may be tumor independent. We predict that effective antiangiogenic therapies will block a tumor's ability to augment angiogenesis initiation rather than subsequent neovessel growth.

Failure of secretin to stimulate gastrin release and adenylate cyclase activity in gastrinoma in vitro.

It has been hypothesized that secretin may act directly on gastrinoma through the adenylate cyclase system to cause stimulation of gastrin release. We studied gastrinoma cells in vitro to determine whether secretin would stimulate gastrin release directly and whether the gastrinoma cell membrane had a functional secretin receptor adenylate cyclase system. Fresh tumor was prepared in cell suspensions containing 1.5 X 10(6) viable cells and incubated for 2 hours with either 2 mM CaCl2 alone (control) or 2 mM CaCL2 and 0.025 U/ml secretin. The gastrin content of the cells in each incubation chamber and the medium were determined by radioimmunoassay and results were expressed as mean gastrin pg/microgram protein +/- SD. Under basal conditions the cellular gastrin content was 39.9 +/- 6.4 (control) compared with 16.7 +/- 2.1 (secretin). After 2 hours of incubation, cellular gastrin content increased in both groups: 68.5 +/- 11.9 (control) to 68.3 +/- 5.5 (secretin). However, the percent of gastrin released into the medium during incubation decreased by one half in both groups (control 37.3% +/- 4.0% to 22.2% +/- 3.0%; secretin 42.8% +/- 7.0% to 18.9% +/- 1.8%). Adenylate cyclase activity was assessed by measuring cAMP generation in fresh-frozen gastrinoma and cultured gastrinoma cell membranes. Isoproterenol (10(-5) M), PGE1 (10(-4) M), and GppNHp (guanine nucleotide) (10(-5) M) caused fivefold to 25-fold increases in cAMP generation. Secretin did not stimulate adenylate cyclase activity above basal (21.73 +/- 4.07 and 2.29 +/- 1.2 pmol cAMP/mg protein/min) for frozen and cultured gastrinoma, respectively. Secretin failed to stimulate gastrin release and adenylate cyclase in vitro. This suggests that secretin-stimulated gastrin release in vivo may not be due to a direct effect of secretin on the gastrinoma.

Treatment of nonendocrine gastrointestinal disorders with octreotide acetate.

Somatostatin and its longer-acting analog, octreotide acetate, can be used effectively for the treatment of nonendocrine gastrointestinal disorders. Octreotide has been shown to decrease pancreatic fistula output by suppressing exocrine pancreatic function. We believe that octreotide acetate may be useful to prophylaxis against the development of pancreatic fistulas following pancreatic resection and may reduce the enzymatic and volume output of established pancreatic fistulas. We also have shown that administration of octreotide acetate 2 hours before a high carbohydrate test meal reduces gut peptide levels, which increase following meal ingestion in patients with the dumping syndrome. Reduction of circulating peptides in these patients may slow gut motility and improve glucose regulation, thus, providing relief of postvagotomy dumping symptoms.

Nonanatomic hepatic resection for secondary hepatic tumors with special reference to hemostatic technique.

The techniques for resection of hepatic tumors have traditionally been based on lobar anatomy. The morbidity and mortality associated with hepatic resection have been most closely correlated with intraoperative blood loss. The results of 37 hepatic resections for secondary neoplasms were retrospectively reviewed. This group included 18 patients who underwent anatomic resections and 19 patients who underwent nonanatomic resections. The nonanatomic group experienced significantly less blood loss, shorter operating times, shorter hospital stays, and no significant difference in long-term survival. A positive relationship between blood loss and postoperative complications is demonstrated for the combined groups. These results support the use of nonanatomic resection whenever feasible for secondary hepatic tumors. We describe the technique for nonanatomic hepatic resection for metastatic lesions, with special emphasis on hemostatic techniques.

Long-term efficacy of octreotide in the treatment of Zollinger-Ellison syndrome.

Nine patients with Zollinger-Ellison syndrome were treated with octreotide acetate (100 micrograms delivered subcutaneously three times daily) and followed up for 1 to 48 months. Serum gastrin levels were obtained at predetermined intervals. All patients had elevated baseline fasting gastrin levels (greater than 150 ng/L [greater than 150 pg/mL]). One month after administration of octreotide, gastrin levels were in the reference range for five (62%) of eight patients, and a mean gastrin suppression rate of 76% was achieved (ie, values were a mean of 76% less than baseline values). One year after administration of octreotide, five (71%) of seven evaluable patients had gastrin levels of less than 200 ng/L (200 pg/mL), and the mean gastrin suppression rate was more than 80% for these seven patients. During the second year, control at these levels was maintained in four patients; one patient continued to have controlled levels for 42 months. Complete symptomatic response occurred in seven patients (78%), and partial response in two patients (22%). All six patients with diarrhea before treatment were cured of it. Octreotide acetate provides efficacious long-term suppression of elevated gastrin levels and excellent symptomatic relief in patients with Zollinger-Ellison syndrome.

The mechanism of hormone-sensitive breast cancer progression on antiestrogen therapy. Implications for treatment and protocol planning.

Fifteen patients whose tumors progressed while they received tamoxifen citrate therapy were studied by serial determinations of serum levels of estrone (E1), estradiol (E2), and dehydroepiandrosterone (DHEA) obtained during progression after withdrawal from tamoxifen therapy and total endocrine ablation or suppression. Discontinuation of tamoxifen therapy resulted in reductions of DHEA, E1, and E2 levels by 44%, 49%, and 42%, respectively. Ablation or suppression reduced sex steroids to minimal levels and produced responses in all patients. Elevations of DHEA, E1, and E2 could be provoked by readministering tamoxifen to hypophysectomized and oophorectomized, but not adrenalectomized, patients, indicating that the adrenal gland is the source of these sex steroids. We conclude that tamoxifen stimulates adrenal production of DHEA, which is aromatized to E1 and E2. Buildup of E1 and E2 overwhelms the competitive binding of tamoxifen to the estrogen receptor, resulting in tumor progression.

Evaluation of bupivacaine nerve blocks in the modification of pain and pulmonary function changes after thoracotomy.

Thirty-one patients undergoing thoracotomy were prospectively randomized to receive (1) no nerve blocks (n = 12), (2) placement of percutaneous catheters for intermittent nerve blocks with bupivacaine (Marcaine) (n = 10), or (3) bupivacaine nerve blocks intraoperatively (n = 8). One patient refused postoperative evaluation and was not included in this study. All patients received similar preoperative, intraoperative, and postoperative medications. Comparison of preoperative and postoperative arterial blood gases, assessments of pain and alertness, and chest roentgenograms showed no statistical advantage for any group. Analgesic requirements and pulmonary functions (functional residual capacity, tidal volume, minute ventilation peak flow, or forced expiratory volume) did not differ among the groups. Statistically significant differences were seen in mean respiratory rate and forced vital capacity. These differences, however, indicate that bupivacaine either by intraoperative use or by intermittent percutaneous administration did not improve postoperative increases in respiratory rate or decreases in forced vital capacity.

Thoracoabdominal-median sternotomy for resection of primary adrenal carcinoma extending into inferior vena cava and hepatic vein.

A technique utilizing a combined thoracoabdominal-median sternotomy approach is described for stimulatenous resection of a large adrenal cancer occluding the inferior vena cava and extending into the left hepatic vein. This technique is useful in removint an extensive extension of a renal or adrenal cancer into the inferior vena cava.

Detection of human chorionic gonadotropin in fresh and formalin-fixed testicular tumor tissue. Comparison of sensitivity of immunoperoxidase to radioimmunoassay.

Immunmocytochemical techniques for the detection of human chorionic gonadotropin (HCG), such as immunoperoxidase or immunofluorescence, allow detection but not quantification of this hormone. A modification of standard radioimmunoassay (RIA) techniques allows utilization of tissue slurries and yields quantitative data on tissue HCG levels. Tissue slurry RIAs and indirect immunoperoxidase were performed on fresh and formalin-fixed tissue specimens from 15 patients with nonseminomatous testicular tumors. HCG detectability by RIA or immunoperoxidase was markedly decreased by formalin fixation. Tissue specimens with high levels of HCG in the fresh state yielded positive immunoperoxidase and RIA results, while specimens with minimally elevated HCG levels tended to be negative. Results of tissue slurry RIAs correlated well with immunoperoxidase results. The detection of HCG in fixed tissue appears to underestimate the amount of HCG present in fresh specimens. Therefore, a negative RIA on formalin-fixed tissue slurry or negative immunoperoxidase of the formalin-fixed, paraffin-embedded tissue does not rule out the absence of HCG in fresh tissue.