Virtual endoscopy is a useful device for training and preoperative planning of transsphenoidal endoscopic pituitary surgery.

Virtual endoscopy (vE) allows simulated three-dimensional (3-D) visualisation of anatomical structures by computerised reconstruction of radiological images. The aim of this study was to evaluate the feasibility of vE and its potential benefits for endoscopic transsphenoidal pituitary surgery. vE was realised using a commercially available ray-casting software plugin of a picture archiving and communications system (PACS). For this study, the vE system was enhanced with volume segmentation, transparency and cutting tools. The data for vE were derived from high resolution computed tomography (CT) scans of 22 patients with pituitary pathology (20 pituitary adenomas, 2 Rathke's cleft cysts) preoperatively. Anatomic structures were identified on vE images and compared with the intraoperative endoscopic views. The simulated 3-D vE images were found to be comparable to the intraoperative endoscopic anatomy in terms of distortion and angle of view. vE was found to be particularly useful for the preoperative depiction of 1) the nasal anatomy and its variations for choosing the side of the approach, 2) the sphenoid sinus septae and chambers for improved intraoperative orientation, 3) the transparent 3-D simulated visualisation of the pituitary gland, tumour and adjacent anatomic structures in relation to the sphenoid sinus landmarks for planning the opening of the sellar floor. We conclude that vE harbours the potential to become a valuable tool in endoscopic pituitary surgery for training purposes and preoperative planning. Furthermore, vE may add to the safety of interventions in case of anatomic variations.

Water-soluble poly-(L-glutamic acid)-Gly-camptothecin conjugates enhance camptothecin stability and efficacy in vivo.

The therapeutic efficacy of 20(s)-camptothecin (CPT) is limited in humans by the instability of the active lactone form due to preferential binding of the carboxylate to serum albumin and by difficulty in formulation. Formation of an ester bond with an amino acid via the hydroxyl group at carbon 20 of CPT stabilizes the lactone. Linking CPT to a high molecular weight (MW) anionic polymer enhances solubility and improves distribution to the tumor through enhanced permeability and retention (EPR effect). Poly-(L-glutamic acid) (PG) is an anionic homo-polymer that can theoretically bind one molecule of a drug via the gamma carboxylic acid of each monomeric subunit. It has been used to make a water-soluble PG-paclitaxel conjugate currently in Phase II clinical trials that contains 37% paclitaxel by weight and is administered in a 10 min infusion without pre-medication. We evaluated the anti-tumor activity of PG conjugates of CPT after a single intraperitoneal injection using subcutaneous murine B-16 melanoma tumor growth as an indicator. Interposition of a glycine (gly) linker allowed CPT loading up to 50% w/w on the polymer. Increasing the PG MW from 33 to 49 kDa enhanced the efficacy without altering the maximum tolerated dose (MTD). In athymic mice bearing ectopic human colon or lung tumors, efficacy was enhanced compared to free camptothecin. Thus, as with paclitaxel, conjugation of CPT to PG enhanced pharmaceutical properties and preclinical efficacy.

A quantitative model for the dynamics of serum prostate-specific antigen as a marker for cancerous growth: an explanation for a medical anomaly.

Prostate-specific antigen (PSA) is an enzyme produced by both normal and cancerous prostate epithelial cells. Although PSA is the most widely used serum marker to detect and follow patients with prostatic adenocarcinoma, there are certain anomalies in the values of serum levels of PSA that are not understood. We developed a mathematical model for the dynamics of serum levels of PSA as a function of the tumor volume. Our model results show good agreement with experimental observations and provide an explanation for the existence of significant prostatic tumor mass despite a low-serum PSA. This result can be very useful in enhancing the use of serum PSA levels as a marker for cancer growth.

Experimental models and methods in antibody targeting of renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is incurable and there are few treatment options that assure even a short prolongation in survival. It is the most common malignancy of the adult kidney and accounts for approximately 12,000 deaths per year (1). Due to a lack of diagnostic markers for early detection and the infrequency of notable symptoms early in the disease process, about one third of patients present with known metastatic disease. However, the staging of RCC is an inaccurate science and in 40% of those patients where a nephrectomy is the treatment choice for presumed organ-confined disease, metastases become evident within about 1 yr (2). For reasons that are poorly understood, metastatic RCC has remained relatively resistant to chemotherapy, biological response modifiers and cellular immunotherapy-although, glimpses of encouragement have appeared in numerous studies. These are discussed in great depth throughout the accompanying chapters.

Intermittent androgen suppression in the LuCaP 23.12 prostate cancer xenograft model.

BACKGROUND: Intermittent androgen suppression (IAS) has been proposed as a method of delaying the onset of androgen-independent growth in prostate cancer. While several pilot studies have demonstrated the feasibility of such a treatment, no study to date has defined the effect of IAS on survival. METHODS: We developed an IAS protocol for mice bearing the LuCaP 23.12 human prostate cancer xenograft, with each cycle consisting of 1 week of androgen replacement with a testosterone pellet followed by 3 weeks of androgen withdrawal. Mice that responded to castration with a 40% or greater decrease in serum prostate-specific antigen (PSA) were randomized to treatment with either continuous androgen suppression (CAS) or IAS. Serum PSA, tumor volume, and overall survival were monitored. RESULTS: A total of 75 mice met the randomization criteria. There was no significant difference of survival between animals treated with CAS or IAS (185 vs. 239 days, P = 0.1835). Serum PSA showed evidence of cycling with hormonal manipulation. No cycling was noted in tumor volume. CONCLUSIONS: IAS is not associated with a decrease in survival compared to CAS, yet in patients may offer quality-of-life improvements. Further studies of IAS in the setting of Institutional Review Board (IRB) approved clinical trials should be encouraged. Prostate 43:63-70, 2000. Published 2000 Wiley-Liss, Inc.

Sicherheit und Vertraglichkeit von GnRH-Agonisten in der Gynakologie.

GnRH agonist (GnRHa) administered for 6 months leads to an effective desensitisation of the pituitary and hypoestrogenism without exerting a particular effect on the whole metabolism. At the end of the first month's a suppression of the serum estradiol levels are achieved, the level of LH and FSH decline in the hypogonadotropic range. No negative influence on the lipid metabolism after administration of GnRH agonist has been observed. The balance of HDL/LDL does not change during the treatment. There were neither any negative changes in the liver metabolism, kidney function nor in the electrolyte values. In anaemic premenopausal women, for example due to serious menstrual problems, a normalisation of the haemoglobin concentration is obtainable already after a 12-week treatment. With regard to the hemostatis system a significant reduction of the procoagulant activity, fibrin turnover rate and a significant improvement of fibrinolytic activity can be observed under a GnRHa therapy. Although the use of GnRHa leads without doubt to a drastic reduction in the uterus blood flow there are no signs that this also leads to a change in the cerebral arteries blood flow. Menstrual bleeding occurs on average 3 months after the last injection of an GnRHa depot injection; with daily injection or nasal spray 3 to 4 weeks earlier. Theoretical considerations as well as the world-wide use as part of the infertility treatment--in some countries more than 90% of all IVF-cycles are performed using GnRH--,contradict the fact that GnRHa cause a teratogenic effect. Domineering undesirable side-effects during a treatment with GnRH can be traced back almost exclusively to the effective hormonal deprivation. In this context it is remarkable which percentage patients complain about trouble of this spectrum before GnRHa treatment is initiated. The chronicle reduction of the sexual hormone level leads without a doubt to a reduction of bone mineral density. The clinical relevance is furthermore a matter of controversial discussion. Prevention measures can be undertaken through an add-back therapy. This can also be of help in the case of vegetative side-effects caused by a decrease in sexual hormones. The question arises to what extent effective non hormonal add-back therapies are at disposal in the treatment of sexual hormone related malignant tumours. Also men with testosterone deprivation can suffer from distinctive hot flushes, sleeping disturbances and depression which requires some kind of relief in order to maintain an acceptable quality of life.

Development of new biotin/streptavidin reagents for pretargeting.

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.

Biotin reagents for antibody pretargeting. 3. Synthesis, radioiodination, and evaluation of biotinylated starburst dendrimers.

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.

Study of free and complexed prostate-specific antigen in mice bearing human prostate cancer xenografts.

BACKGROUND: Our objective was to evaluate five preclinical prostate cancer (CaP) xenograft models to determine whether (1) prostate-specific antigen (PSA) formed complexes in murine serum, (2) the percentage of free PSA (f-PSA) was characteristic of a given xenograft line, and (3) the percentage of f-PSA was similar to that in the patient at time of tumor harvest. Our fourth objective was to identify which murine serpin(s) bind(s) to PSA in vivo. METHODS: Xenografts were established from metastatic foci. The percentage of f-PSA, and total PSA (t-PSA) in serum of animals bearing CaP xenografts was determined by immunoassay. Size exclusion high-performance liquid chromatography and Western blots were used to evaluate the presence of PSA complexes in murine serum. Edman degradation was used to determine the N-terminal sequence of complexed proteins. RESULTS: PSA was detected as both free and complexed forms in murine serum from all mice bearing the CaP xenografts. Three xenografts (related sublines) produced PSA that resulted in low mean percentages of f-PSA (1.9-6.4%). In sera from the other two xenografts, the mean percentages of f-PSA were high (>25%); patient sera, where available at time of tumor acquisition, were in agreement. Western blots showed that murine protease inhibitors formed complexes with PSA. Edman degradation yielded a sequence with 80% homology over 15 amino acids with that of murine alpha1-protease inhibitor (alpha1-PI). CONCLUSIONS: Our data have shown that the majority of PSA secreted by these CaP xenografts complexes in murine serum with a protease inhibitor with high homology to murine alpha1-PI and that the percentage of f-PSA is a characteristic of each xenograft line tested, which is in agreement with patient values at time of tumor harvest. These CaP xenografts offer opportunities for study of human PSA biology and phenomenology.

[What is the value of combined surgical-hormonal therapy of endometriosis management?]. / Was leistet die kombinierte chirurgisch-hormonelle Therapie zum Management der Endometriose?

Medical oestrogen suppressive therapy has to be considered as an important principle in the management of endometriosis. In the last years GnRHa became the "gold standard" as pre or postoperative measures before/after surgical intervention. We analyzed the data of 198 patients, most of them with recurrent endometriosis histologically confirmed during first-look laparoscopy. Patients were treated in a prospective, multicentre phase III study with the six months GnRHa leuprorelinacetate depot (LAD) followed by a second-look laparoscopy for precise assessment of therapeutic effects. In all stages of endometriosis a 35% reduction of the r-AFS-score compared to the baseline could be achieved due to surgical intervention during first-look laparoscopy with a further improvement of 64% after GnRHa-therapy and surgery during second-look laparoscopy. Two subgroups of patients (24 vs. 45) could be analyzed according to the time of second-look laparoscopy (< or = 30 days vs. > or = 60 days after last injection) showing a comparable r-AFS-score reduction. Both, superficial lesions and deep infiltrating nodules, endometriomas, peritoneal implants and obliterated cul de sac could successfully be treated through the combined medical-surgical approach. Pre- or postoperative therapy with a GnRHa facilitates surgical excision of the implants, a subtle adhesiolysis and often complete removal of all visible lesions. In a high percentage of patients, including advanced stages of the disease, a preservation of ovarian tissue to ensure the childbearing potential could be achieved by minimal-invasive techniques. These results can be claimed as the prerequisites for long-term relief of endometriosis complaints and encouraging pregnancy rates in endometriosis related infertility. This confirms great clinical benefit of the combined medical-surgical approach for the treatment of this enigmatic disease.

Streptavidin in antibody pretargeting. 2. Evaluation Of methods for decreasing localization of streptavidin to kidney while retaining its tumor binding capacity.

An investigation has been conducted to determine if the kidney localization of recombinant streptavidin can be decreased to improve its characteristics in pretargeting protocols. Three different methods of accomplishing this were evaluated. The first method, blocking kidney uptake with a preadministration of recombinant streptavidin in which biotin occupied all of the binding sites, was unsuccessful. In a second method, l-lysine administration was used to block kidney localization. This method worked well, decreasing the concentration to 29% of the unmodified amount at 8 h postinjection. However, this method suffered from a requirement for constant infusion of lysine during the period of observation. A third method, use of succinylated recombinant streptavidin, was found to be the best approach. Succinylation of streptavidin was readily accomplished with very good protein recovery. With the succinylated streptavidin, the kidney concentration was only 14% of that of nonmodified streptavidin at 4 h postinjection. While these results demonstrated that the concentration of streptavidin could be decreased in the kidney, it was important to assess whether the tumor colocalization of streptavidin with biotinylated antibody was affected under those conditions. As part of our continuing investigation of pretargeting, a new water-solubilized biotinidase-stabilized biotinylation reagent was prepared. Using that reagent in a pretargeting experiment, an equivalent quantity of succinylated recombinant streptavidin as biotinylated antibody Fab' was localized in a tumor xenograft model. In that experiment, the kidney concentration was decreased to less than 10% of that obtained with unmodified recombinant streptavidin at 24 h postinjection. The results of our investigation have demonstrated that succinylation of streptavidin improves its distribution characteristics for pretargeting applications. The fact that succinylated streptavidin has no specific tissue localization should allow its use as a carrier of radioactivity in "two-step" pretargeting protocols.

LNCaP produces both putative zymogen and inactive, free form of prostate-specific antigen.

BACKGROUND: Our objective was to investigate the presence of prostate specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) mRNA and protein in prostate cancer cell lines, and the complexing characteristics of expressed PSA. METHODS: RT-PCR, immunohistochemistry, and Western blots were employed. Trypsin treatment of PSA was performed to establish the possible presence of an activatable form of PSA. RESULTS: ACT mRNA and protein were detected in LNCaP, PC-3, and DU 145 by RT-PCR and by immunohistochemistry, respectively. Only LNCaP cells were positive for PSA mRNA and protein. LNCaP expressed approximately 30% active PSA, approximately 40% putative zymogen form of PSA, and approximately 30% stably inactive PSA. CONCLUSIONS: We have shown that the majority of PSA expressed by LNCaP cells is present in free, noncomplexed forms in the conditioned media. A portion (40%) can be activated by trypsin, while the rest is stably inactive PSA. LNCaP cells may serve as a source of the "unreactive" PSA present in prostate cancer patients' serum.

Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins.

In this investigation, a comparison of wild type recombinant streptavidin (r-SAv) with two genetically engineered mutant r-SAv proteins was undertaken. The investigation also included a comparison of the r-SAv with two streptavidin (SAv) proteins from commercial sources. In vitro characterization of the SAv proteins was conducted by HPLC, SDS-PAGE, IEF, and electrospray mass spectral analyses. All SAv proteins studied appeared to be a single species by size exclusion chromatography (HPLC) and SDS-PAGE analyses, but multiple species were noted in the IEF and MS analyses. In vivo comparisons of the SAv proteins were accomplished with dual isotope-labeled SAv in athymic mice. In an initial experiment, tissue localization of r-[131I]SAv directly radiolabeled using chloramine-T was compared with r-SAv radiolabeled with the N-hydroxysuccinimidyl p-iodobenzoate conjugate ([125I]-PIB), a radioiodination reagent that has been shown to result in iodine-labeled proteins which are stable to in vivo deiodination. The data obtained indicated that there is little difference in the distribution (except kidney localization) when r-SAv labeled by the two methods. Data obtained from comparison of r-[131I]SAv with a disulfide-stabilized r-SAv mutant (r-SAv-H127C), a C-terminal cysteine-containing r-SAv mutant (r-[125I]SAv-S139C), and two 125I-labeled SAv proteins obtained from commercial sources indicated that their distributions were quite similar, except the kidney concentrations were generally lower than that of r-[131I]SAv. On the basis of the similar distributions of the SAv proteins studied, it appears that the r-SAv mutants may be interchanged for the (wild type) r-SAv in pretargeting studies. Further, the similarity of distributions with two commercially available SAv proteins suggests that the results obtained in our studies and those of other groups may be directly compared (with consideration of animal model, sacrifice time, etc.).

A novel monoclonal antibody 107-1A4 with high prostate specificity: generation, characterization of antigen expression, and targeting of human prostate cancer xenografts.

Monoclonal antibodies with high specificity for prostate tissue are of interest for prostate cancer research and treatment. Reactivity and specificity of a new murine monoclonal antibody, 107-1A4, was assessed by immunohistochemistry, ELISA and indirect immunofluorescence (IDIF). 107-1A4 stained all normal and malignant prostate tissue specimens while reactivity to non-prostate tissue was limited to the tubules of the normal kidney and renal cell carcinoma. Twenty two human cell lines were included in the reactivity survey; only the immunogen prostate cancer line LNCaP reacted with 107-1A4. Seminal plasma proteins PSA, PAP, PSMA, and PSP-94 were determined not to be the 107-1A4 antigen.

Cross-reactivity of ten anti-prostate-specific antigen monoclonal antibodies with human glandular kallikrein.

OBJECTIVES: Prostate-specific antigen (PSA) is commonly used as a marker for prostate disease. Prostate epithelium expresses both PSA and human glandular kallikrein (hK2) proteins, which share 80% sequence homology. The immunologic cross-reactivity of these two proteins could potentially interfere with determination of PSA levels in diagnoses of prostate cancer. We set out to determine the extent of this cross-reactivity for a panel of 10 anti-PSA monoclonal antibodies (mAbs). METHODS: Enzyme-linked immunosorbent assay (ELISA), sandwich assays, and western transfer techniques were used to assess the PSA/hK2 cross-reactivity of the anti-PSA mAbs. RESULTS: We did not detect the hK2 protein with any of the 10 anti-PSA mAbs under western transfer conditions. In ELISA experiments, 8 of 10 mAbs exhibited hK2 cross-reactivity under certain conditions. However, no combination of mAbs tested in sandwich assays exhibited a signal in hK2 cross-reactivity experiments greater than 0.1% of the PSA signal. CONCLUSIONS: We have evaluated 10 anti-PSA mAbs and determined that despite the 80% homology between PSA and hK2 proteins, cross-reactivity with hK2 by these antibodies would not significantly affect the determination of PSA levels by means of sandwich assays.

Long-term follow-up on the treatment of endometriosis with the GnRH-agonist buserelinacetate. Long-term follow-up data (up to 98 months) of 42 patients with endometriosis who were treated with GnRH-agonist buserelinacetate (Suprecur), were evaluated in respect of recurrence of pain symptoms and pregnancy outcome.

OBJECTIVE: In our previous study, 119 patients with histologically confirmed endometriosis underwent a 'three-step' therapy between 1987 and 1989, where surgical removal of endometriosis was followed by a 6 month treatment with 3 x 300 microgram buserelinacetate daily intranasally and a second look laparoscopy or laparotomy with removal of residuals. Long-term follow-up data in respect of recurrence of symptoms and pregnancy outcome were investigated. STUDY-DESIGN: Long-term follow-up data of 42 out of 119 treated patients on the post-treatment effect were obtained using a special questionnaire. Recurrence of dysmenorrhea, dyspareunia and pelvic pain was defined as recurrence of disease. The follow-up period was up to 98 months with a median time of 82.5 months. RESULTS: Out of the 42 patients, 23 complained of infertility. Fourteen out of these 23 patients became pregnant during the follow-up period, resulting in 23 pregnancies with 18 newborns, 4 miscarriages and one ectopic pregnancy. Ten patients conceived spontaneously, stimulation program became necessary in the rest of patients. Twenty-eight of the 42 patients complained recurrence of symptoms with median first onset at 10.7 months. Improvement on quality of life and subjective conditions were reported by 30 patients. CONCLUSIONS: Our study suggests that the 'three-step' therapy of endometriosis with GnRH-agonist buserelinacetate leads to a significant improvement on the quality of life and well being in the majority of the patients and to a high pregnancy rate. This treatment represents a favourable approach in the management of endometriosis.

Uterine leiomyomata and sterility: therapy with gonadotropin-releasing hormone agonists and leiomyomectomy.

The aim of this study was to obtain data about the pregnancy rate in patients with uterine leiomyomata after treatment with gonadotropin-releasing hormone (GnRH) agonists followed by myomectomy. Between 1987 and 1993, 61 patients with uterine leiomyomata and sterility underwent 6 months' GnRH agonist treatment, in part with a surgical intervention. Sixty-two per cent of the patients suffered from concomitant endometriosis. After hormonal therapy 41 patients underwent a myomectomy. According to sonographic and clinical criteria, there was no indication for the enucleation of the leiomyomata for the remaining 20 patients. Owing to the combined therapy, consisting of primary treatment of uterine leiomyomata with GnRH agonists, followed by surgical intervention, 25 patients (41%) suffering from long-term sterility (average 4 years) became pregnant. An early abortion occurred in only three cases (12%). No patient who underwent a myomectomy developed new myomata during the following pregnancy. Four patients suffering from a single leiomyoma became pregnant within the first 3 months after myomectomy, all of them conceiving spontaneously. Considering the high rate of spontaneous conceptions and the low abortion and complication rates during pregnancy, the combined therapy of GnRH agonists followed by myomectomy represents a major step forwards in the effective treatment of sterility in patients with uterine leiomyomata.

Microvascular endothelium of human tumor xenografts expresses mouse (= host) CD31.

BACKGROUND: Human malignant tumors grown as xenografts in immunocompromised animals have been used extensively to study tumor growth and tumor response to therapy. The endothelium functions as an effective barrier between the intravascular space and the tumor cells. In a previous study we used species-specific monoclonal antibodies against endothelial cell adhesion molecules to demonstrate the host origin of the endothelium in xenotransplanted pancreatic islet grafts [Am J Pathol 1995;146:1397-1405]. We now investigated in this study whether the vascular endothelium of different xenografted human malignant tumors expresses mouse (= host)- or human (= graft)-specific CD31 (platelet endothelial cell adhesion molecule, PECAM-1) adhesion molecules. METHODS AND RESULTS: Cultured human prostate, kidney, and colon cancer cells (passages 15-17) were transplanted subcutaneously into 8-week-old athymic nude mice and removed after another 8 weeks. The avidin biotin peroxidase method was utilized on frozen sections to demonstrate that the endothelium of the vasculature of all three human xenografts expressed mouse (= host)-specific CD31, but not human (= graft)-specific CD31. CONCLUSION: The presence between the intravascular space and the human tumor cells of a mouse-derived endothelium, expressing mouse-specific antigens, needs to be taken into careful consideration when evaluating results of antitumor therapies in these animal models. This caveat pertains particularly to the study of novel cell- or tissue-specific treatment modalities, such as antibody-targeted drugs, toxins or radionuclides, 'immuno'-liposomes, or tumor vaccines.

Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab' colocalization with recombinant streptavidin and avidin.

An evaluation of the use of a biotinylated monoclonal antibody Fab' fragment in tumor pretargeting was conducted. As a model system, tumor colocalization of avidin or recombinant streptavidin (r-streptavidin) and the biotinylated Fab' fragment (Fab'-S-biotin) of A6H, an antirenal cell carcinoma antibody, was evaluated in athymic mice bearing human renal cell carcinoma xenografts. A new water soluble sulfhydryl reactive biotinylation reagent, N-(13-N-maleimdo-4, 7,10-trioxatridecanyl)-biotinamide, was synthesized and used for biotinylation of Fab'. A biodistribution of ChT-labeled A6H Fab'-S-biotin was conducted. Data from that distribution indicated that the Fab'-S-biotin localized well (i.e. 28% ID/g at 24 h) to human tumor xenografts in athymic mice. Subsequently, a biodistribution study involving pretargeting radioiodinated A6H Fab'-S-biotin to tumor xenografts, followed by administration of r-streptavidin at 4 or 20 h, was conducted. Specific colocalization of r-streptavidin to tumors containing the A6H Fab'-S-biotin was evident from the data obtained. In a similar biodistribution study, specific colocalization of avidin to tumors pretargeted with A6H Fab'-S-biotin was also observed. The avidin used in the study was radioiodinated with the N-hydroxysuccinimidyl ester of p-[125I]iodobenzoate ([125I]PIB-NHS). Very low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the tumor. To further show that specific colocalization within the tumor xenografts had occurred with biotinylated A6H Fab', radioiodinated avidin and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated Fab' present. At 20 h postinjection, only small differences in the blood and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodistributions must have been due to the presence of Fab'-S-biotin. Calculations were conducted to estimate how much r-streptavidin (as a molar ratio) was colocalized. From the data obtained it was estimated that 36-61% of the tumor-localized Fab'-S-biotin molecules were bound with r-streptavidin and 4-23% bound with avidin, under the conditions studied.

Cell proliferation and apoptosis during prostatic tumor xenograft involution and regrowth after castration.

The biological mechanisms involved in androgen-dependent and -independent prostate cancer growth after castration were analyzed in the LuCaP 23.1 human prostate cancer xenograft model. Athymic mice (n = 82) bearing LuCaP 23.1 xenograft were castrated and tumors were harvested at different time points from day 0 to day 112 post castration. In each group of mice, tumor growth rate (TGR), serum PSA concentration, percentage of tumor cells incorporating bromodeoxyuridine (BUdR index), percentage of apoptotic tumor cells assessed by morphological analysis (apoptotic index), and presence of apoptosis-related DNA "ladder" were analyzed. Castration induced a significant decrease in TGR and serum PSA from day 1 to day 7, and a progressive increase in the 2 parameters from day 14 to day 112, heralding androgen-independent tumor relapse. Meanwhile the BUdR and apoptotic indexes varied as follows after castration: an increase was noted for both at day 3, a significant increase in apoptotic index with a decrease in BUdR index from day 5 to day 14, and a progressive decrease in apoptotic index while BUdR index remained at 50% of the pre-castration value from day 28 to day 112. DNA ladder was present sparsely in tumors grown in non-castrated hosts, universally present in tumors from day 1 to day 28 post castration, and frequent in tumors from day 56 to 112. Castration-induced effects in LuCaP 23.1 tumors were characterized by an increase in number of apoptotic cells and a decrease in proliferative activity. The androgen-independent tumor relapse after castration was associated with a low apoptotic index with no increase in proliferative activity.