Radioimmunoguided breast surgery using radiolabeled antibody NR-LU-10 FAB: a pilot study.

AIM AND BACKGROUND: Radioimmunoguided surgery using radiolabeled NR-LU-10 Fab was evaluated as a method of intraoperative breast cancer detection. METHODS: Breast cancer patients were injected intravenously with 125I (74 MBq) labeled NR-LU-10 Fab (5 mg) and then underwent tumor excision 2, 4 or 7 days later, during which time the gamma detector probe was used to evaluate the primary tumor for evidence of radioactive uptake. RESULTS: Intraoperative probing revealed tumor localization in 7 of 10 patients (70%). Gamma probe counts of the excised tumor were elevated in all patients, although high counts in surrounding non-malignant tissue obscured the ability to detect the tumor in vivo in 3 patients. One patient with bilateral breast cancer was found to have a separate focus of occult tumor in each breast using the gamma detector probe. CONCLUSIONS: Radiolabeled NR-LU-10 Fab possesses favorable pharmokinetics and tumor-binding ability as a targeting agent. However, binding to non-malignant tissue limits its role in the intraoperative evaluation of tumor margins in breast cancer patients. Its role in other malignancies should be explored.

18F-labeled fluorodeoxyglucose positron emission tomography-guided surgery for recurrent colorectal cancer: a feasibility study.

OBJECTIVE: Positron emission tomography (PET) scanning is an accepted diagnostic tool for the detection of colorectal cancer (CRC). The purpose of this study was to determine whether diagnostic information offered by preoperative PET scan could be used to detect disease intraoperatively using beta and gamma handheld probes. METHODS: Two studies were carried out. First, tumor "phantoms" were created using 62 microCi fluorodeoxyglucose (FDG) in a saline-filled basin. Gamma and beta handheld probes were used to determine detection characteristics with respect to probe type, distance from source, and isotope half-life. In a second study, probes were used intraoperatively to detect tumor in 10 patients with recurrent colorectal cancer as determined by preoperative PET scan. Counts relative to background were determined for each probe as was histopathologic correlation with probe-positive tissue. RESULTS: Phantom studies documented that FDG detection by each probe was nonlinearly related to source proximity and half-life. In human subjects, abnormal findings on preoperative PET studies were detected by both probes with tumor:normal ratios of 1.6 (beta) and 1.5 (gamma). All probe-positive tissue was histologically confirmed to be recurrent colorectal cancer. CONCLUSIONS: Intraoperative detection of CRC using an FDG source and beta and gamma probes correlates with preoperative PET. With further improvements in probe technology, successful differentiation of normal and tumor tissue as shown here may allow for more precise localization and directed resection.

Correlative Whole-Body FDG-PET and Intraoperative Gamma Detection of FDG Distribution in Colorectal Cancer.

Purpose: 18F-Fluorodeoxyglucose-positron emission tomography (FDG-PET) is the superior imaging modality for detection of primary and recurrent colorectal cancer compared to magnetic resonance imaging (MRI) or computerized tomography (CT). We investigated the feasibility of developing intraoperative procedures for detection of FDG in tumor deposits in order to assist the surgeon in achieving an optimal reduction of tumor burden.Procedures: Fourteen patients (45-83 years of age) were scanned using FDG-PET followed by Gamma Detection Probe evaluation at laparotomy. One patient did not have a pre-operative FDG-PET scan. The collimated detector probe contained a CdZnTe crystal (7mm diameter x 2mm thick). We used a lower window setting of 200 KeV and an open upper window setting. Fasted patients were given an IV bolus of FDG (4.0-5.7 mCi) 15-20 minutes prior to preparation for surgery. Catheterization and the diuretic Lasix were used to remove FDG activity from the bladder. The time from FDG injection to intraoperative GDP data acquisition varied from 58-110 minutes.Results: In all patients, the GDP detected background activity in normal tissues (aorta, colon, liver, kidney, abdominal wall, mesentery, and urinary bladder). The GDP correctly identified single or multiple tumor foci in 13/14 patients as noted by an audible signal from the control unit (3 S.D. above counts obtained from normal tissues). These tumor foci corresponded to regions of high FDG uptake as seen on FDG-PET scans. The one case that the GDP did not localize was a recurrent mucin pseudomyxoma-producing tumor (acellular, mucinous deposits). Ex vivo GDP evaluations demonstrated significant tumor:normal adjacent tissue activity (audible signals in 6/6 tumor samples tested).Conclusions: These data demonstrate that tumors identified from pre-operative whole-body PET scans can be localized during surgery utilizing a gamma probe detector and FDG.

Biodistribution and localization of radiolabeled NR-LU-10 Fab fragment in human breast cancer xenografts.

Radioimmunodetection, which takes advantage of tumor-specific or tumor-associated radio-labeled monoclonal antibodies or other biologic molecules to diagnose the extent of disease in cancer patients, has been of limited use in studies to date in patients with breast cancer. The difficulty is in finding an antibody that is both sensitive and specific enough to localize in breast tumors. This study undertook immunohistochemical and in vivo evaluation of tumor localization and biodistribution of NR-LU-10 Fab (antibody fragment) in breast tumors to determine its ability to bind selectively to malignant tissue. NR-LU-10 Fab recognizes a pancarcinoma glycoprotein antigen found on tumors of epithelial cell origin. NR-LU-10 Fab reacted with 6/6 (100%) breast cancer cell lines and 14/16 (87.5%) breast tumors with varying degrees of immunostaining intensities. Athymic mice bearing ZR-75-1 breast cancer xenografts were injected with 125I-labeled NR-LU-10 Fab (12 microg/5 microCi) and sacrificed at fixed time intervals. These studies demonstrated the highest tumor uptake of labeled Fab at 12 h postinjection (4.58+/-1.59% of injected dose/gram [% ID/g] of tissue); this gradually decreased to 0.13+/-0.05% ID/g of tissue by 72 h postinjection of the radiolabeled Fab. Biolocalization to normal tissues was as predicted for a Fab fragment; i.e., initially high in clearance organs (kidney), followed by rapid clearance over the 72-h test period. NR-LU-10 Fab displays adequate breast tumor localization with minimal biolocalization to normal tissues, thus supporting its potential use in radioimmunoscintigraphy and the RIGS system (radioimmunoguided surgery).

Lysine reduces renal accumulation of radioactivity associated with injection of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid-CC49 Fab radioimmunoconjugate.

The high uptake and prolonged renal retention of monoclonal antibody fragments that are conjugated with radiometal chelates precludes their routine clinical use due to high background counts, which may hinder detection of nearby lesions and/or cause renal radiotoxicity. We report on the potential use of Lys as a pharmacological agent to enhance renal excretion of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid CC49 Fab ([177Lu]CC49 Fab) radioimmunoconjugate. The monoclonal antibody portion of this complex is directed toward the tumor-associated glycoprotein-72 antigen. Lys was administered to female BALB/c mice by i.p. injections. [177Lu]CC49 Fab bolus injections were given by the i.v. route. Results of our investigations showed that: (a) kidney radioactivity concentrations were inversely related to Lys dose. The optimal dose (50 mg/mouse) evoked a 3-fold reduction in kidney counts; (b) Lys was most effective when injected 15 min before, or at the same time as, [177Lu]CC49 Fab; (c) the renal effect was both rapid (3-fold decrease at 15 min after injection) and prolonged (4-fold decrease at 24 h after injection); (d) a single Lys dose decreased total body radioactivity by > 2.5-fold; (e) urine excretion of radioactivity was enhanced in Lys-treated mice. High pressure liquid chromatographic analyses using a GF-250 column showed that a large fraction of this urine radioactivity coeluted with a [177Lu]CC49 Fab injection standard. We conclude that Lys enhances the urinary excretion of radioactivity associated with [177Lu]CC49 Fab. These observations warrant further study with regard to the use of amino acids or their derivatives as pharmacological agents to enhance the urinary excretion of small-molecule radioimmunoconjugates.

Combined immunohistochemical and autoradiographic analyses of antigen/antibody interactions in tumor xenograft models.

A study was conducted to establish optimal conditions which would allow for the simultaneous localization of a carcinoma antigen and its complementary radiolabeled antibody. Immunoperoxidase staining was used to identify the tumor distribution of antigen, while tissue localization of the radiolabeled antibody was identified by autoradiography. The tumor associated glycoprotein-72 (TAG-72) antigen and the high affinity murine monoclonal antibody, CC49 IgG were used as the model antigen/antibody pair. Athymic female mice bearing either CX-1 or LS-174T human colorectal adenocarcinoma xenografts were used as animal/tumor test systems. Experimental mice each received a bolus intravenous injection of the CC49 antibody which was labeled with 125I (specific activity, 0.17 to 0.26 microCi/microgram). Control mice were given a bolus injection of MOPC-21 IgG monoclonal antibody (tumor irrelevant antibody) which was also radiolabeled with 125I (specific activity, 0.24 to 0.35 microCi/microgram). At 24 hours postinjection, all tumors removed, counted for radioactivity, and fixed in formalin. The avidin/biotin immunoperoxidase complex technique was used to identify TAG-72 antigenic sites on slide-mounted tissue sections. Nonradiolabeled CC49 IgG (0.5 micrograms/ml) was used as the specific antigen binding primary antibody in the immunostaining procedures. Nonradiolabeled MOPC-21 IgG (0.5 micrograms/ml) served as the negative control. Immunohistochemically stained tissue sections were coated with photographic emulsion and processed for autoradiographic localization of 125I-CC49 or 125I-MOPC-21. After an optimal exposure time of 6 days, slides were processed and examined under a light microscope. Results of the biolocalization experiment revealed that the % of injected dose/gram of 125I-CC49 in both LS-174T and CX-1 tumors (30.4 +/- 5.2% and 20.6 +/- 5.4%, respectively) were significantly greater (p greater than 0.01) than those for 125I-MOPC-21 (4.9 +/- 0.5% and 5.1 +/- 0.7%, respectively). In both tumor lines from mice injected with 125I-CC49, dense clusters of silver grains were found over those regions which were positive for TAG-72 immunoreactivity. These dual-labeled structures were also found in contact with, or in close proximity to the microvasculature. Tumors from mice which were injected with the control radioconjugate showed a random distribution of silver grains within stromal tissue but no specific localization to TAG-72 positive regions. We conclude that intravenously administered 125I-CC49 IgG localizes specifically to antigen-containing sites in the LS-174T and CX-1 tumor models. The methods described herein should serve as useful tools for the direct study of antigen-antibody interactions in tumor biology.

Age-, sex-, and gonadal steroid-related changes in immunoreactive substance P in the rat anterior pituitary gland.

The interrelationship of anterior pituitary lobe (AP) immunoreactive substance P (I-SP) concentrations with age, sex, gonadal steroids, and estrous cyclicity in rats was examined. There was no difference between male and female AP I-SP levels at 0.5 month of age. At 2.0 and 5.0 months of age, a sex-linked difference in AP I-SP concentrations was evident, inasmuch as male APs contained approximately 3 and 8 times greater concentrations of I-SP, respectively, than APs from age-matched females. Long term (6 weeks) gonadectomy of adult rats resulted in an increase in I-SP concentrations in female APs and a decrease in the concentrations of the peptide in male APs compared to values in their respective sham-operated controls. Treatment of gonadectomized animals for the same length of time with gonadal steroid-filled Silastic capsule implants resulted in qualitatively identical responses in males and females; that is, estradiol benzoate decreased and 5 alpha-dihydrotestosterone propionate increased AP I-SP levels compared to the respective control values in castrates. Testosterone propionate treatment had no effect on AP I-SP levels compared with the respective gonadectomy control values. Short term (8 days) gonadectomy of adult males did not affect the AP concentration of I-SP. Likewise, gonadectomy of adult females was ineffective in altering the AP I-SP concentration compared with concentrations in females on diestrous day 1, diestrous day 2, proestrous, or estrous stages of the vaginal cycle. These data suggest that gonadal steroids are physiologically important in the regulation of I-SP concentrations in the AP. We hypothesize that I-SP is indigenous to the AP and that gonadal steroids act directly at the level of the AP to affect the synthesis and/or release of the peptide.

Changes in hypothalamic and pituitary content of immunoreactive alpha-melanocyte-stimulating hormone during the gestational and postpartum periods in the rat.

alpha-Melanocyte-stimulating hormone (alpha-MSH) was measured in the mediobasal hypothalamus (MH), median eminence (ME), preoptic-suprachiasmatic area (POA-SCN), anterior (AL), and posterior lobes (PL) of the pituitary gland during the gestational and postpartum periods in the rat. The content of alpha-MSH in the MH and POA-SCN compared to estrous levels was lower during the later days of gestation and decreased further in the MH during lactation in association with the elevated plasma prolactin (Prl). Distinct increases in the ME content of alpha-MSH compared to estrous levels occurred on Days 8 and 12 of the gestational period and Day 14 of the postpartum period. A significant increase in PL content of alpha-MSH compared to Days 5-11 and 17-20 occurred on Day 4 of gestation, and no significant changes were detected in the AP concentration of alpha-MSH throughout the period studied. In vitro, PLs and ALs from females on Day 4 of gestation secreted more alpha-MSH into the incubation medium than tissues from animals on Day 20. These results suggest that alpha-MSH of both brain and pituitary origin may play a role in mediating some of the physiological changes which occur during pregnancy and lactation.

Hypothalamic control of prolactin secretion during the perinatal period in the rat.

The development of hypothalamic control of PRL secretion in the late prenatal and early postnatal periods in the rat was studied by employing a static system for incubation of pituitaries. PRL was detected in the incubation medium after incubating fetal pituitaries as early as day 18. A gradual increase in the amount of hormone released into the medium occurred during development, with the greatest change occurring between days 20 and 21 prenatally. A progressive increase in the pituitary content of PRL occurred during development. The percentage of PRL released from the gland was higher pre- (19-39%) than postnatally (17%). Hypothalamic extracts from fetuses and neonates stimulated the secretion of PRL when coincubated with pituitaries from animals of the same age. However, extracts from 1-day-old neonates inhibited PRL secretion from adult male hemipituitaries. Extracts prepared from adult male hypothalami stimulated PRL secretion from neonatal pituitaries from rats at 1 day of age, as did cerebral cortical extracts from adults, but not 1-day-old, rats. TRH significantly stimulated PRL secretion in vitro from pituitaries of donors as early as day 19 of the fetal period. The response of the pituitaries to this peptide diminished by day 8 postnatally. Immunoneutralization of hypothalamic TRH significantly decreased but did not abolish the PRL-releasing activity of hypothalamic extracts, whereas this procedure had no effect on the GH-releasing activity of the extracts. The dopamine receptor agonist apomorphine (10(-5) M) inhibited PRL secretion in vitro on day 19 of the fetal period and postnatally starting on day 1. The response to apomorphine increased with advancing age. The results suggest that a combination of factors contribute to maintaining high circulating PRL levels during the late fetal and early neonatal periods. These include high rates of PRL release by the pituitary and a relative insensitivity of the pituitary to dopamine in the face of high sensitivity to PRL-releasing factors such as TRH.

Partial characterization of immunoreactive substance P in the rat pituitary gland.

Two distinct carboxy-terminus-directed anti-substance P (SP) sera (R-1C and R-6G) were used to characterize immunoreactive SP (I-SP) in acetic acid extracts of anterior pituitary (AP) and posterior pituitary (PP) glands of adult male rats. The tissue concentrations of I-SP measured by R-1C and R-6G were comparable. The contents of I-SP were 600-1150 pg/AP and 25-52 pg/PP. I-luteinizing hormone releasing hormone and I-somatostatin (I-SOM) were undetectable in AP extracts, but PP extracts contained the equivalents of 325-785 pg I-SOM/gland. Serial dilutions of AP and PP extracts produced displacement curves with both SP antisera that were parallel to the respective synthetic SP standard and hypothalamic extract displacement curves. Gel filtrations of AP and PP extracts on a Sephadex G-25 column produced I-SP peaks eluting in the same fractions as synthetic SP and hypothalamic I-SP. However, the AP I-SP profile also revealed a side peak migrating between the void volume and the major I-SP peak. Neither immunoreactive species in the AP extract were eliminated when eluted with 6.0 M guanidine HCl, a strong denaturing agent. In vitro incubation of paired anterior hemipituitaries for 30 min in the presence of a 56 mM K+ concentration resulted in a significant (p less than .0001), 25-fold increase in the release of I-SP into the incubation medium above the mean control value. Radiofrequency lesions placed in the median eminence-arcuate region of male rats caused a significant (p less than .001) reduction of I-SP in both the AP and PP. These reductions were inversely related to the plasma prolactin values. The elevation in plasma prolactin was taken as an index of completeness of lesions. We conclude that: 1) the rat pituitary contains I-SP as assessed by its immunologic and chromatographic behavior, 2) K+ depolarization is a potent stimulator of the release of AP I-SP in vitro, 3) the ME-arcuate region is important for the maintenance of pituitary I-SP levels in the rat.

The effect and possible mode of action of alpha-melanocyte-stimulating hormone on gonadotropin release in the ovariectomized rat: an in vivo and in vitro analysis.

This study was designed to examine the possible effects of alpha MSH on gonadotropin release. Injection of alpha MSH into the third ventricle of the brain and sampling at 5, 10, 15, 30, and 60 min postinjection produced a significant lowering of plasma LH, but not of FSH, in conscious ovariectomized (OVX) rats. The minimum effective dose was 1 microgram. This procedure did not affect plasma levels of LH or FSH in estradiol benzoate-primed, OVX rats. Multiple blood sampling every 10 min before and after intraventricular injection of alpha MSH (2 micrograms) produced a significant reduction in the area under the secretion curve of LH. Intravenous injection of alpha MSH had little effect; however, a slight lowering of plasma LH occurred in OVX rats after a 50-micrograms dose. The effect of alpha MSH on LH release was blocked by pretreatment of the animals with alpha-methyl-paratyrosine, an inhibitor of catecholamine synthesis, as well as by pretreatment with an iv injection of spiroperidol, a dopamine receptor blocker. The peptide failed to alter basal or K+-stimulated LHRH release from median eminence fragments of OVX rats incubated in vitro. Alpha MSH had no effect on LHRH-induced LH release in vivo and failed to alter the release of FSH and LH from hemipituitaries of OVX rats or dispersed cells from OVX, estradiol benzoate-primed rats in vitro. It is concluded that alpha MSH exerts an inhibitory effect on LH release by an action on the hypothalamus, probably via activation of the tuberoinfundibular dopaminergic system.

Effect of unilateral orchidectomy on plasma FSH concentration: evidence for a direct neural connection between testes and CNS.

The effect of unilateral orchidectomy on plasma FSH, LH and LHRH content in the right and left half of the median eminence-arcuate nucleus region was examined in adult rats. Following right-sided orchidectomy, plasma FSH began to rise but a significant increase was not observed until 1 week after the operation. No significant changes were observed in LH values. LHRH content in the ipsilateral median eminence-arcuate region showed a significant increase only when the median eminence was removed together with the arcuate nucleus, but not when it was removed separately, which suggests that the increase in LHRH content was attributable to an increased LHRH content of the ipsilateral arcuate nucleus. Radiofrequency lesions placed unilaterally in the same side of the dorsal anterior hypothalamic area as the hemiorchidectomy partially blocked the selective FSH release following unilateral orchidectomy, while contralateral or sham lesions had no effect. These results suggest that the gonad has a direct neural connection with the hypothalamus, which is involved in selective FSH release following hemiorchidectomy.

Development of hypothalamic control of growth hormone secretion in the rat.

The development of hypothalamic control of GH in the late prenatal and early postnatal periods in the rat was studied by employing a static system for the incubation of pituitaries. The basal secretion of GH into the medium after a 3-h incubation period showed a gradual increase from day 18 prenatally to day 1 postnatally. This was followed by a gradual decline in GH release on postnatal days 5 and 8. There was a sustained rise in the total pituitary GH content from prenatal day 18 to postnatal day 8. The percentage of the total GH that was released into the medium was high from fetal pituitaries and lower from neonatal pituitaries. TRH (100 ng/ml) stimulated GH secretion starting on prenatal day 21. This TRH effect persisted through day 8 postnatally. Hypothalamic extracts from fetuses and neonates stimulated the secretion of GH when coincubated with pituitaries of the same age and with adult male rat pituitaries. Similarly, adult male rat hypothalamic extract stimulated the secretion of GH from pituitaries of 1-day-old neonates. Pronase treatment of neonatal hypothalamic extract completely abolished its stimulatory effect on GH release. Incubation of 1-day postnatal pituitaries with cerebral cortical extract obtained from neonates of the same age did not alter the secretion of GH; however, cerebral cortical extract from adult males did cause a significant stimulation of GH release from the neonatal pituitaries. Somatostatin (100 ng/ml) failed to inhibit GH release by pituitaries until day 5 postnatally, but a 10-fold increase in the concentration of somatostatin significantly inhibited GH secretion from pituitaries of rats as early as day 21 prenatally. Coincubation of hypothalamic extract with the high concentration of somatostatin significantly attenuated the effect of the extract in stimulating GH release from pituitaries of 1-day-old rats. The results suggest that the high circulating levels of GH during the late prenatal and early neonatal periods are maintained by a combination of factors including the release of a hypothalamic peptidergic GH-releasing factor, the relative insensitivity of the pituitary to somatostatin, and changes in the relative size of storage vs. releasable pools of GH during development.

Substance P immunoreactivity in the anterior pituitary gland of the guinea pig.

The aim of this study was to demonstrate the presence of Substance P (SP) immunoreactive cells within the anterior pituitary gland of the adult guinea pig. By utilizing the PAP technique of Sternberger, immunoreactive SP-containing cells were visualized in all animals studied. These cells were most dense in the ventral portion of the gland. When adjacent tissue sections were incubated with anti-SP serum and anti-rat TSH serum, it was observed that most, if not all, the SP immunoreactive cells examined also exhibited TSH immunoreactivity. However, not all TSH immunoreactive cells contained SP immunoreactivity. Immunoabsorption controls indicated that the immunohistochemical staining reactions were specific for both SP and TSH. The results of this study indicate that immunoreactive SP is present in the guinea pig anterior pituitary and that this peptide is localized within TSH immunoreactive cells.

Annual variations in plasma levels of testosterone and luteininzing hormone in the laboratory male mongrel dog.

Blood samples, drawn every 15 days (September 1975-September 1976) from four laboratory-housed male mongrel dogs, were assayed by radioimmunoassay for levels of testosterone and LH in the plasma. The mean plasma concentrations of testosterone remained relatively constant for most of the year with the exception of a significant rise in late August and early September. Mean plasma levels of LH showed a cyclic pattern throughout the year which could be represented by a cosine function curve. However, this cyclic pattern of LH was not accompanied by cyclic changes in plasma levels of testosterone and there was no relationship between these two hormones during the period of 1 year. As the cyclic pattern of LH was altered, the plasma level of testosterone began to rise and reached its highest concentration. Since this alteration of the LH cycle occurred before the increased concentrations of testosterone, and since there was no relationship between these two hormones for the period of a year, we have concluded that there may be another hormone(s) involved which either alters the sensitivity of the canine testis to LH or alters the Lh synthesis/release mechanism of the pituitary gland.

Plasma concentrations of testosterone and LH in the male dog.

Blood samples were withdrawn every 20 min from 3 conscious intact and 2 castrated mature males during non-consecutive periods of 12 h during the light and dark phases of the lighting schedule (intact dogs) and of 11 h during the light period (castrated dogs). In the intact dogs testosterone concentrations ranged from 0.4 to 6.0 ng/ml over the 24-h period. LH concentrations varied from 0.2 to 12.0 ng/ml. In all animals, LH peaks were clearly followed, after about 50 min, by corresponding testosterone peaks, but no diurnal rhythm could be established. LH concentrations in the castrated dogs were high (9.8 +/- 2.7 (s.e.m.) ng/ml), and still showed an episodic pattern in spite of the undetectable plasma testosterone levels.